Pieter Janssen
email: pieter.janssen@med.kuleuven.be

Hans Pottel

Rita Vos

Jan Tack

Endogenously released opioids mediate meal-induced gastric relaxation via peripheral mu-opioid receptors

Keywords: opioids, barostat, food intake, satiation, intragastric pressure, methylnaltrexone, naloxone

Endogenously released opioids mediate meal-induced gastric relaxation via peripheral mu-opioid receptors.

Introduction

During food intake smooth muscle of the stomach relaxes so that the stomach can accommodate the sometimes large quantities of food we eat without major increase in intragastric pressure (IGP) 1 . This reflex relaxation, also referred to as gastric accommodation is likely to play a part in the control of food intake 2,3 .

Delta-, kappa-and mu-opioid receptors are found throughout the enteric and central nervous system and influence gastrointestinal motility and sensation [4][5][6][7] . Only a limited number of studies indicate a complex role for opioids in the control of proximal stomach motility: in different barostat studies, opioid receptor agonists morphine and remifentanil have both increased and decreased gastric tone [8][9][10] while the centrally-acting mu-opioid receptor antagonist naloxone inhibited meal-induced gastric accommodation 11 . Also the role of opioids in the regulation of food intake is complex: in general opioid agonists enhance feeding and opioid antagonists decrease feeding 12,13 but these effects are dependent for example on the treatment duration and whether normal weight, low weight (anorexia patients) or obese volunteers/patients are selected 5 .

The various and sometimes opposing findings with opioid agonists and antagonists in search for the role of opioids in the control of food intake and gastric motility could be explained by the fact that central and peripheral opioid receptors mediate various and sometimes opposing effects e.g. in the control of intestinal motility in rats 14 , gastric relaxation to a meal in dogs 15 or gastric emptying in humans 16 . Recently, the peripherally restricted and selective mu-opioid receptor antagonist methylnaltrexone was approved for the treatment of opioid-induced constipation 17 . In order to study the role of peripheral mu-opioid receptors in the regulation of gastric tone and food intake we investigated the effect of methylnaltrexone on gastric sensitivity, gastric compliance and gastric relaxation after a meal in a barostat study.

Recently the barostat has been criticized: the inflated balloon distends the proximal stomach, might exaggerate gastric accommodation and hamper physiological responses to food intake 18- 20 . We therefore developed a new technique to assess gastric accommodation during food intake by measuring the IGP 21 . In the present study we investigated the effect of methylnaltrexone in a barostat study of which the protocol was identical to the one in which we previously investigated the effect of naloxone on gastric sensorimotor function 11 . In order to confirm the barostat findings we performed separate experiments that investigated the effect of both naloxone and methylnaltrexone on gastric tone and accommodation to a meal by measuring the IGP during food intake.

Methods

Ethical approval

All study procedures were approved by the Ethics Committee of the Leuven University Hospital, Belgium. Written, informed consent was obtained from all subjects, and studies conformed to the Declaration of Helsinki.

Study subjects

A total of 23 healthy volunteers (HV's, 12 men, age: 28±2 years, body mass index: 21.7±0.4 kg m -2 ) participated in the studies. None of the HV's had symptoms or a history of gastrointestinal disease, other significant diseases, psychological disorders or drug allergies; none were taking any medication or had a drug history. HV's were asked to refrain from alcohol, tea and coffee at least 12 hours before participation, and refrain from smoking cigarettes at least 1 hour before the start of the experiment. Study populations differed between the different experiments (isobaric barostat distension to determine compliance and sensitivity, barostat experiment to determine gastric accommodation after meal intake, IGP during food intake) although some subjects participated in more than one experiment. Between 2 experiments with the same subject at least 1 week washout was respected.

Barostat studies

After an overnight fast of at least 12 h, a double lumen polyvinyl tube (Salem sump tube 14 Ch; Sherwood Medical, Petit Rechain, Belgium) with an adherent polyethylene bag (maximal volume 1200 ml; 17 cm maximal diameter) was introduced through the mouth and secured to the subject's chin with adhesive tape. The correct position of the bag in the gastric fundus was checked fluoroscopically. The polyvinyl tube was then connected to a programmable barostat device (Synectics Visceral Stimulator, Stockholm, Sweden). To unfold the bag, it was inflated with a fixed volume of 300 ml for 2 min with the subject in a recumbent position and again deflated completely. The subjects were then positioned in a comfortable sitting position with the knees bent (80°) and the trunk upright in a specifically designed bed. After a 30-min adaptation period, the minimal distending pressure (MDP) was determined by increasing the intra-bag pressure with 1 mmHg every 3 min, until a volume of 30 ml or more was reached 22 .

Hereafter either compliance and sensitivity or gastric accommodation to a meal was determined.

Compliance and sensitivity to distension 13 HV's (HV's, 7 men, age: 31±2 years, body mass index: 21.9±0.6 kg m -2 ) were enrolled for this experiment. 15 minutes after treatment with either methylnaltrexone [12 mg subcutaneous injection (0.6 ml)] or placebo (subcutaneous injection of 0.6 ml saline) isobaric distensions were performed in stepwise increments of 2 mmHg starting from MDP, each lasting for 2 min, while the corresponding intragastric volume was recorded. Subjects were instructed to score their perception of upper abdominal sensations at the end of every distending step. They used a global graphic rating scale that combined verbal descriptors on a scale graded 0-6 22 . The end point of this distension sequence was established at an intra-bag volume of 1500 ml, or when the subjects reported discomfort or pain (score 5 or 6). Hereafter, the volunteers could leave the hospital.

For each 2 minute isobaric distending period, the intragastric volume was calculated by averaging the recording. The thresholds for perception and discomfort were computed after the experiments by analyzing the perception score corresponding to each distension step.

Perception threshold was defined as the lowest pressure relative to MDP that evoked a perception score of 1 or more, and the corresponding volume. Discomfort threshold was defined as the lowest pressure relative to MDP and the corresponding volume that provoked a score of 5 or more. Pressure-volume and pressure-perception curves were obtained from the stepwise distensions and fitted with a linear regression model as previously described 22,23 .

Gastric compliance was calculated as the slope and the intercept of the pressure-volume curve obtained during the isobaric distensions.

Gastric accommodation to a meal 11 HV's (HV's, 6 men, age: 29±2 years, body mass index: 22.1±0.7 kg m -2 ) were enrolled for this experiment. After positioning of the barostat bag, the pressure level was set at MDP + 2 mmHg. The intra-bag volume at this pressure was recorded for 15 min, and then either placebo or methylnaltrexone was subcutaneously administered.

Intra-bag volume was continuously monitored for 90 min, after the first 30 min, a standardized liquid meal (200 ml, 300 kcal; 13% proteins, 48% carbohydrates, 39% lipids; Nutridrink; Nutricia, Bornem, Belgium) was administered. Barostat measurements continued for 60 min after the start of meal ingestion.

To evaluate gastric tone before and after administration of the meal, mean intra-balloon volume was calculated over consecutive 5-min intervals of the measurement.

Statistical analysis Paired Student's t-tests were used to assess whether the mean values of two parameters differ between both treatment groups. Gastric accommodation was compared using a repeated measured ANOVA as the mean area under the curve in the hour after the liquid meal intake. All data are presented as the mean ± standard error of the mean (SEM). A p-value < 0.05 was considered to be statistically significant (NS: not significant).

Intragastric pressure measurement during nutrient drink infusion 11 HV's (HV's, 5 men, age: 28±2 years, body mass index: 22.2±0.6 kg m -2 ) were enrolled for this experiment. After an overnight fast of at least 12 h, a high-resolution solid-state manometer system (36 channels, 1 cm in between each channel, Manoscan 360, Sierra Scientific Instruments, Los Angeles, USA, Manoview analysis software v2.0.1) was positioned through the nose so that at least 1 sensor was positioned in the lower esophageal sphincter (LES; detected as a clearly elevated pressure zone compared to oral and aboral areas), while IGP was measured using the average pressure of the first 5 pressure channels that were clearly positioned below the LES or the pressure area influenced by the LES (in most cases this was approximately 3-8 cm under the LES). A second infusion catheter (Flocare, Nutricia, Bornem, Belgium) was positioned in the stomach through the mouth. The tip of the infusion catheter was positioned approximately 5 cm under the LES and its position was verified by fluoroscopy.

The catheters were fixed to the subjects chin and the HV's were asked to take a comfortable position in a bed. Volunteers were randomly assigned to 1 of 3 treatment regimens: 1) placebo methylnaltrexone (0.6 ml saline s.c.) and placebo naloxone (intravenous saline 100 ml h -1 after a 1 ml bolus injection)

2) methylnaltrexone [12 mg subcutaneous injection (0.6 ml)] with placebo naloxone 3) placebo methylnaltrexone with naloxone (infusion rate of 20 µg kg -1 h -1 after a 0.4 mg bolus injection; Narcan®, Bristol-Myers Squibb Pharma, Braine-l'Alleud, Belgium).

Treatment was applied after a stabilization period of at least 15 minutes and 15 minutes hereafter nutrient drink (Nutridrink, Nutricia, Zoetmeer, The Netherlands; 630 KJ, 6 g proteins, 18.4 g carbohydrates, and 5.8 g lipids per 100 ml) was infused directly in the stomach at a constant speed of 60 ml min -1 determined by an automated system using a peristaltic pump (Infusomat SpaceP; Braun Medical, Diegem, Belgium). Starting 5 minutes before nutrient drink ingestion HV's were asked to score their satiation at 1-minute intervals, using a graphic rating scale that combines verbal descriptors on a scale graded of 0-5 (1, threshold; 5, maximum satiation). Intragastric infusion was stopped as soon as a score of 5 was reached on their satiation scores, hereafter the experiment was terminated.

The original data were imported from the recording software to an Excel (Microsoft Corporation, CA, USA) spreadsheet. We were primarily interested in slow IGP changes that could reflect changes in gastric muscle tone. Therefore, and in order to avoid influence from movement artifacts as well as artifacts caused by coughing, sneezing, moving or swallowing a moving minimum was calculated per channel from the original data (minimum value over 30 seconds of original data) followed by a moving median (median value over 120 seconds of the moving minimum data). Data were analyzed and represented per minute as the average value of the 5 measurement channels that were clearly positioned below the LES as described above.

Statistical analysis In order to compare the results a linear mixed model of a completely randomized design with repeated measures and factors treatment and time was constructed.

All analyses were performed with SAS 9.2 (SAS Institute Inc., Cary, NC, USA). When the data before the start of the nutrient drink infusion was modeled, no treatment main effect (p=0.081) or treatment-by-time interaction effect (p=0.98) was observed. Therefore, and in order to compare the effect of a treatment on the IGP during nutrient drink infusion a baseline value (calculated as the average pressure in the last 5 minutes before nutrient drink infusion started) was subtracted from the data before comparison. Data are presented as mean±S.E.M. Relations between the IGP increase from nadir and the corresponding satiation score increases were examined by correlation analysis (p<0.05 is considered significant).

Results

Influence of methylnaltrexone on gastric compliance and sensitivity to distension, measured with the barostat

The mean MDP on the day of saline and methylnaltrexone administration was not significantly different (6.7±0.3 vs. 6.9±0.3 mmHg; NS). During isobaric distensions, gastric compliance remained unaltered after saline and methylnaltrexone administration (43.6±4.9 vs. 43.6±4.8 ml mmHg -1 ; NS; figure 1A). Also, methylnaltrexone administration had no significant influence on the pressure-perception curves (figure 1B; AUC 5.4±0.3 vs. 5.4±0.4; NS). The pressures needed to induce first perception (10.0±0.6 vs. 9.9±0.6 mmHg; NS) or discomfort (16.0±0.6 vs. 17.0±0.4 mmHg; NS) and the corresponding intra-balloon volumes (respectively 246±33 vs. 223±22 and 514±45 vs. 490±48 ml; NS) did not differ between saline and methylnaltrexone.

Influence of methylnaltrexone on gastric accommodation to a meal, measured with the barostat Preprandial intragastric volumes were similar in both saline and methylnaltrexone condition (143 ± 16 vs. 124 ± 23 ml; NS). Ingestion of the meal caused an immediate relaxation of the proximal stomach in all subjects, reflected by an increase in the balloon volume (figure 2).

During the administration of methylnaltrexone, the gastric accommodation was significantly inhibited (p<0.01). Influence of methylnaltrexone and naloxone on intragastric pressure during nutrient drink infusion, measured with high resolution manometry No significant treatment effect (p=0.081) or treatment-by-time effect (p=0.98) could be detected before the start of the nutrient drink infusion. When comparing the IGP during nutrient drink infusion a clear treatment effect (p<0.005), time effect (p<0.0001) and treatmentby-time effect (p<0.01) could be observed. Significant differences between treatments at individual time points are shown in figure 3.

HV's scored maximal satiation after 1124±107, 979±96 and 958±84 ml nutrient drink infused (for placebo, naloxone and methylnaltrexone treatment respectively; P<0.05 between placebo and methylnaltrexone treatment).

Discussion

The role of opioids in the control of proximal stomach motility is seemingly complex: it has been shown that the centrally acting mu-opioid receptor agonists morphine and remifentanil both increased and decreased gastric tone depending on the study protocol and center [8][9][10] . In a previous barostat study we performed in which the used methods are same as the present study the centrally acting mu-opioid receptor antagonist naloxone had no effect on basal gastric tone but inhibited meal-induced gastric accommodation, although the significance of the latter finding was borderline 11 . In the present study we showed that although methylnaltrexone did not affect basal gastric tone, the effect of methylnaltrexone on gastric accommodation was more pronounced than that of naloxone.

The effect on gastric accommodation was assessed using the barostat technique and an alternative technique to assess gastric accommodation. Indeed, although the barostat is still considered the gold standard to assess gastric tone and accommodation, the nature of this measurement is regarded as unphysiological: the inflated balloon distends the proximal stomach, might exaggerate gastric accommodation and hampers physiologic responses to food intake [18][19][20] . We therefore developed a new technique to assess gastric relaxation during food intake in a more physiological manner, by measuring the IGP during food intake 21 . Indeed, it is generally accepted that the tone of the stomach muscles decrease during food intake, to allow storage of the meal without a rise in IGP 24,25 . Using this principle to assess gastric accommodation, we found that during nutrient drink ingestion, the IGP decreased rapidly to gradually increase thereafter while nutrient drink ingestion is continued. When we impaired gastric accommodation by treating the volunteers with the nitric oxide synthase inhibitor N G - monomethyl-l-arginine the IGP during nutrient drink ingestion was significantly increased, supporting the hypothesis that IGP reflects gastric accommodation 21 . In the present study, barostat and IGP measurements both confirmed impaired accommodation after methylnaltrexone, indicating that both are valid to assess gastric accommodation.

Despite the shortcomings of the barostat technique it is at present the only technique that can assess the effect of a treatment on sensation to distension and compliance. After methylnaltrexone treatment stepwise increase in intraballoon pressure did not change perception as compared to placebo. Moreover pressures needed to induce first perception and discomfort were not influenced by methylnaltrexone. These findings argue against a role for peripheral mu-opioid receptor activation in the control of sensitivity to gastric distension and confirm our previous findings with naloxone 11 . As argued in the latter paper our findings do not exclude efficacy of exogenously applied agonists in the control of visceral sensitivity. Indeed, opioids are well-known for their antinociceptive effects in somatic but also visceral pain 26 . A possible limitation of our experimental design is that we only distended until the volunteers reported discomfort while it could be argued that endogenous opioids are only released when truly painful sensations are elicited. On the other hand, in similar barostat studies we performed with functional dyspeptic patients the same range of pressure values was used to show that these patients were hypersensitive to gastric distension 22,27 .

Gastric accommodation is a reflex relaxation of the stomach muscles. In short, this reflex consists of vagal afferent neurons that synapse in the nucleus tractus solitarii (NTS) and the dorsal motor nucleus of the vagus and can activate vagal efferent neurons that in turn activate inhibitory motor neurons (mainly nitrergic in nature) in the enteric nervous system of the stomach to finally decrease the gastric muscle tone 28 . Endogenous opioids might be involved in several ways, hereafter we will discuss 2 options:

-Mu-opioid receptors have been identified on nerve terminals in the myenteric plexus of the stomach 29 . In rats it has been shown that (mu-) opioid receptor agonists inhibit vagal stimulation-induced acetylcholine release in isolated, vascular perfused stomachs 30 . Although endogenous release of opioids in the enteric nervous system during the gastric accommodation reflex has not been described it is plausible that opioid receptors on acetylcholine releasing nerves are activated during the gastric accommodation reflex inhibiting acetylcholine release which could result in a decreased gastric tone. Indeed, in isolated rat stomachs endogenous opioids are released upon vagal nerve stimulation 31 , and enkephalins are likely to be the predominant endogenous opioids that activate neuronal mu opioid receptors in the gut 6 .

-In the medial subnucleus of the tractus solitarii (mNTS) mu-opioid receptors are localized 32 while the presence of endogenous mu receptor agonists as endomorphin-1 and 2 has been described 33 . Herman et al. performed a series of experiments in rats that showed that microinjection of mu opioid receptor agonists in the mNTS reduced the gastric tone while injection of mu-opioid receptor antagonists per se had no effect on the gastric tone. However, esophageal distension-induced gastric tone decrease was blocked by injection of mu-opioid receptor antagonists in the NTS 34 . The authors proposed that upon activation of the vago-vagal reflex pathway that leads to gastric tone decrease, vagal afferents release opioids (possibly endomorphine-2) that inhibit GABA interneurons in the mNTS. Once the influence of GABA signaling is depressed, glutamate released from the vagal afferents can excite second-order NTS neurons and the vago-vagal reflex pathway that inhibits the gastric tone is activated 34 . This hypothesis could well explain our findings: we did not observe an effect of methylnaltrexone on basal gastric tone but the food-induced decrease in gastric tone was blocked by methylnaltrexone.

The question remains why the effect of the peripherally restricted mu-opioid receptor antagonist methylnaltrexone was more pronounced than that of the centrally-acting antagonist naloxone. Both methylnaltrexone and naloxone can reach the NTS 34 , however naloxone can also influence higher brain areas that might cancel the effects it has on the NTS or the enteric nervous system out. Opioids are well-known to inhibit GABA release also in the higher centers of the central nervous system 35 , opioid antagonists such as naloxone could disinhibit GABAergic transmission in higher central nervous system areas and counteract the effects in the NTS, although this remains speculation with regard to the gastric accommodation reflex.

In the present study we also showed that methylnaltrexone significantly increased satiation during food intake, while no significant effect of naloxone could be observed. The apparent difference with our previous study where naloxone significantly enhanced meal-induced satiation 11 , is probably attributable to the relatively small size of the effect of naloxone and the limited sample size. Also in the literature there is some controversy with regard to the role of opioids in the control of food intake: in general opioid agonists enhance feeding and opioid antagonists decrease feeding 12,13 . It has however been shown that this effect is dependent, for example, on the treatment duration and whether normal weight, low weight (anorexia patients) or obese patients are selected 5 . Similar to the effects on proximal stomach motility this complexity can be explained by the fact that different opioid receptors have different effects at the peripherally and at the central level. It has previously been suggested that gastric accommodation is a determinant of satiation both in healthy volunteers and FD patients with weight loss using the barostat, IGP measurements and imaging techniques 2,36,37 . Also in this study impaired gastric accommodation after methylnaltrexone treatment might explain why volunteers drank less.

Methylnaltrexone and naloxone were administered in a different dose and route of administration. Moreover, both drugs have different binding affinities to the mu receptor 38,[START_REF] Holzer | Opioid receptors in the gastrointestinal tract[END_REF] and their pharmacokinetic profiles are different [START_REF] Garnock-Jones | Methylnaltrexone[END_REF][START_REF] Albeck | Quantitative and pharmacokinetic analysis of naloxone in plasma using high-performance liquid chromatography with electrochemical detection and solid-phase extraction[END_REF] . Although direct comparison is difficult we have dosed the drugs as they are prescribed in routine clinical practice 11,17 . We therefore assume that they are similarly effective as mu-opioid receptor antagonists and that observed differences can be attributed to the fact that methylnaltrexone has no described effects centrally while naloxone passes the blood brain barrier.

In conclusion, we have shown that although the peripherally restricted mu-opioid receptor antagonist methylnaltrexone has no effect on gastric sensation to distension or compliance, gastric accommodation to a meal was impaired and feelings of satiation were enhanced. These results indicate that endogenous opioids mediate gastric accommodation and satiation via peripheral mu-opioid receptors. The fact that the effects were less pronounced or absent after treatment with the centrally acting mu-opioid receptor antagonist naloxone indicate that centrally involved mu-opioid receptors may mediate an opposing effect on gastric accommodation.

Abstract

Objective The stomach relaxes upon food intake and thereby provides a reservoir for the ingested meal while keeping the intragastric pressure (IGP) low. We set out to determine whether we could use IGP as a measurement for stomach accommodation during food intake.

Design In fasted healthy volunteers (HVs; n=7-17) and functional dyspeptic patients with unexplained meal-induced dyspeptic symptoms (n=8) a manometer and an infusion catheter were positioned in the proximal stomach. After a stabilization period a nutrient drink was intragastrically infused at 15, 30 and 60 ml min -1 . To investigate the effect of impaired accommodation the effect of N G -monomethyl-l-arginine (L-NMMA) was examined. The volunteers scored satiation until maximum, when the experiment ended. IGP was presented as a change from baseline (mean±SEM) and compared with repeated measures ANOVA.

Results Independent on the ingestion speed, the IGP decreased initially and gradually increased thereafter. HVs scored maximal satiation after 699±62, 809±90 and 997±120 ml nutrient drink infused (15, 30 and 60 ml min -1 respectively; P<0.01). Maximum IGP decrease was 3.4±0.5 mmHg after 205±28 ml, 5.1±0.7 mmHg after 212±46 ml, and 5.2±0.7 mmHg after 296±28 ml infused volume (15, 30 and 60 ml min -1 respectively; NS). Significant correlations were found between IGP and satiation score increase. During L-NMMA infusion IGP was significantly increased while subjects drank significantly less (816±91 ml vs. 1032±71 ml; p<0.005).

Interestingly, the correlation between IGP increase and satiation score increase did not differ after L-NMMA treatment. Patients tolerated less nutrient drink (408±131 vs. 998±61 ml; p<0.0005) while the IGP decrease was significantly decreased (1.7±0.7 vs. 4.2±0.4 mmHg; p<0.01).

Conclusions IGP during nutrient drink ingestion provides a minimally invasive alternative to the barostat for the assessment of gastric accommodation in HVs and patients. These findings furthermore indicate that IGP is a major determinant of satiation. The golden standard to determine gastric accommodation is the gastric barostat ( 6). This technique detects changes in muscle tone by measuring volume changes of an intragastric balloon that is kept at a constant pressure. There are however important drawbacks associated with the technique: the inflated balloon distends the proximal stomach, might exaggerate gastric accommodation and hampers physiologic responses to food intake (7;8). Moreover, muscle tone can only be measured in a limited stomach region and the invasive nature of this technique, which is perceived as uncomfortable and stressful, excludes its application in routine clinical practice. There is a clear need for an alternative technique to assess gastric accommodation during food intake (9).

We recently described a novel technique to measure gastric accommodation during intragastric nutrient drink infusion in rats based on IGP measurements (10). Indeed, IGP is determined by the gastric muscle tone and we demonstrated that increasing or decreasing the gastric tone increased or decreased IGP, respectively. There is also emerging evidence in man that gastric accommodation determines IGP during stomach distension: while in healthy subjects the IGP increase during stomach balloon or air distension is minor or stable and does not increase despite further distension, IGP increase was much more pronounced in patients that underwent vagotomy, patients with Chagas' disease and patients with functional dyspepsia (11-14). Despite these encouraging reports, IGP during gastric distension has never been advanced to standardization and validation for minimally invasive assessment of gastric accommodation in man. kg m -2 ) participated in the studies (some volunteers participated in more than one protocol).

F

None of the HV's had symptoms or a history of gastrointestinal disease, other significant diseases, psychological disorders or drug allergies; none were taking any medication or had any drug history.

Functional dyspeptic (FD) patients: 8 patients were selected from an outpatient clinic pool of patients that presented with unexplained dyspeptic symptoms. Inclusion criteria were the presence of unexplained meal-induced dyspeptic symptoms (bothersome post-prandial fullness or early satiation that prevents the finishing of a regular sized meal at least several times per week for at least the last three months with an onset of symptoms at least six months prior to diagnosis). Organic, systemic or metabolic diseases were excluded as possible explanation for the symptoms by means of upper gastrointestinal endoscopy, routine biochemistry and upper abdominal ultrasound, performed as routine clinical examinations. Exclusion criteria were predominant heartburn, the presence of oesophagitis, gastric atrophy or erosive gastroduodenal lesions on endoscopy, heartburn as a predominant symptom, a history of peptic ulcer, major abdominal surgery, underlying psychiatric illness and the use of non-steroidal anti-inflammatory drugs, steroids or drugs affecting gastric acid secretion.

All participants participated after an overnight fast, furthermore were they asked to refrain from alcohol, tea and coffee at least 12 hours before participation, and to refrain from smoking cigarettes at least 1 hour before the start of the experiment.

Intragastric balloon distension

Upon arrival in the clinic, a double lumen polyvinyl tube (Salem sump tube 14 Ch. Sherwood Medical, Petit Rechain, Belgium) with an adherent polyethylene bag (maximal volume 1200 ml;

17 cm maximal diameter) was introduced through the mouth and secured to the subject's chin with adhesive tape. The polyvinyl tube was then connected to a programmable barostat device (Synectics Visceral Stimulator, Stockholm, Sweden). To unfold the bag, it was slowly inflated with a volume of 300 ml for 2 minutes with the subject in a recumbent position, hereafter the bag was completely deflated. The subjects were then positioned in a comfortable sitting position with the knees bent (80°) and the trunk upright in a specifically designed bed. After a 30-minute stabilization period the subjects were asked to drink 200 ml of either tap water or a nutrient drink (Nutridrink; Nutricia, Bornem, Belgium), 5 minutes later the intragastric balloon was inflated at a rate of 60 ml min -1 . The HV's were randomly allocated to the treatment in a crossover fashion. Subjects were asked to score satiation and abdominal sensation using a graphic rating scale that combined verbal descriptors on a scale graded 0-5 (1=threshold, 5=maximum satiation or discomfort). The balloon was deflated when a score of 5 was reached.

IGP measurement during nutrient drink infusion

2 types of manometry probes were used: to study the influence of intragastric nutrient drink infusion vs. drinking (using different speeds) a custom-made 6-channel solid-state manometer was used with 4 cm in between each pressure channel (Konigsberg Instruments Inc., Probe Model P31-6019DD18, recording software: Polygram for Windows 2.05 (Synectics, Stockhom, Sweden)). For all other experiments a high-resolution solid-state manometer system was used (36 channels, 1 cm in between each channel, Manoscan 360, Sierra Scientific Instruments, Los Angeles, USA, Manoview analysis software v2.0.1). Upon arrival in the clinic the manometer was positioned through the nose so that at least 1 sensor was positioned in the lower esophageal sphincter (LES; detected as a clearly elevated pressure zone compared to oral and aboral areas), while IGP was measured using the sensor 4 cm under the LES (Koningsberg catheter) or as the average pressure of the first 5 pressure channels that were clearly positioned below the LES or the pressure area influenced by the LES (approximately 3-8 cm under the LES (high resolution catheter (see data analysis; figure 1)).

General protocol

In those experiments in which the volunteers had to drink the nutrient drink, only the manometry probe was placed. For the experiments in which the nutrient drink was intragastrically infused a second infusion catheter (Flocare, Nutricia, Bornem, Belgium) was positioned in the stomach through the mouth. The tip of the infusion catheter was positioned approximately 5 cm under the LES and its position was verified by fluoroscopy. From preliminary experiments we found that the position of the infusion catheter (with the tip in the proximal or distal stomach) did not affect the IGP during intragastric nutrient drink infusion (60 ml min -1 ; n=5; results not shown).

The catheter(s) was/were fixed to the subjects chin and the HV's were asked to take place in a chair. In preliminary experiments we found that the position of the volunteer did not influence IGP and satiation indicating that gravitational forces do not influence the results. After a stabilization period of at least 15 minutes, nutrient drink (Nutridrink, Nutricia, Zoetmeer, The Netherlands; 630 KJ, 6 g proteins, 18.4 g carbohydrates, and 5.8 g lipids per 100 ml) was infused directly in the stomach at a constant speed (15, 30 or 60 ml min -1 ) determined by an automated system using a peristaltic pump. When IGP was studied during drinking, a peristaltic pump was used to automatically fill cups (15, 30 or 60 ml min -1 ), while the HV's were asked to empty the cups so they could keep up with the filling rate.

HV's were asked to score their satiation at 1-minute intervals, using a graphic rating scale that combines verbal descriptors on a scale graded of 0-5 (1, threshold; 5, maximum satiation). At 5minute intervals the HV's were asked to fill out a visual analogue scale for 11 epigastric symptoms. Intragastric infusion or drinking was stopped as soon as the HV's scored maximally on one of the 11 epigastric symptoms or when a score of 5 was reached on their satiation scores, hereafter the experiment was terminated.

IGP

The original data was imported from the recording software to excel. We were primarily interested in slow IGP changes that could reflect changes in gastric muscle tone. Therefore, and in order to avoid influence from movement artifacts as well as artifacts caused by coughing, sneezing, moving or swallowing a moving median was calculated per channel from the original data (median value over 1 minute of original data). Per channel, a baseline value was calculated from the moving median data as the average pressure in the last 5 minutes of the stabilization period. Data was presented per minute as the difference of the minimum moving median value in that minute and the baseline value. When the high-resolution manometer was used, data was represented as the average value of the 5 measurement channels that were clearly positioned below the LES as described above. Data was presented as mean±S.E.M. and compared with a two-way repeated measures ANOVA and a Tukey post-test for multiple comparisons when applicable. Relations between the IGP increase from nadir and the corresponding satiation score increases were examined by correlation analysis (P<0.05 is considered significant).

The results in patients were compared with all the data from control experiments in which nutrient drink was intragastrically infused at 60 ml min -1 .

F

Results

IGP during balloon distention

Balloon pressure increased progressively during inflation (figure 2). After the subjects drank water the pressure in the bag increased significantly more as compared to nutrient drink (n=9; p<0.05). Balloon inflation was stopped when subjects scored discomfort at 671±34 ml (after water ingestion) and 714±53 ml (after nutrient drink ingestion; NS).

IGP during intragastric nutrient drink infusion or drinking: effect of speed of ingestion

HV's scored maximal satiation after 699±62, 809±90 and 997±120 ml nutrient drink infused (15, 30 and 60 ml min -1 respectively; p<0.01 between 15 and 60 ml min -1 ; n=17). When the subjects drank the nutrient drink, maximum satiation was reached after 810±85, 823±87 and 1018±119 ml (15, 30 and 60 ml min -1 respectively; p<0.05 between 15 and 60 ml min -1 ; n=13). During infusion or while drinking the IGP initially decreased rapidly, followed by a gradual increase thereafter (figure 3). Maximum IGP decrease during nutrient drink infusion was 3.4±0.5 mmHg after 205±28 ml for 15 ml min -1 , 5.1±0.7 mmHg after 212±46 ml for 30 ml min -1 and 5.2±0.7 mmHg after 296±28 ml for 60 ml min -1 (NS). Maximum IGP decrease during drinking was 5.2±0.8 mmHg after 272±54 ml for 15 ml min -1 , 6.5±1.4 mmHg after 201±31 ml for 30 ml min -1 and 4.8±1.0 mmHg after 231±56 ml for 60 ml min -1 (NS).

Correlation between IGP recovery and satiation scores

A significant correlation was found between the IGP increase from nadir and the corresponding satiation scores in each group (p<0.0001; figure 3B). During nutrient drink infusion the correlation coefficients were not significantly different between infusion speeds, while during drinking, the correlation coefficient at 15 ml min -1 was significantly smaller that when volunteers drank 60 ml min -1 (p<0.05). Both for the infusion and drinking data, the slopes of the linear regressions and the intercepts were not significantly different from each.

Nutrient drink vs. water, viscous saline and a solid meal HV's ingested significantly more water than nutrient drink before maximal satiation was scored (1198±139 vs. 956±92 respectively; p<0.05; n=9). Maximum IGP decrease was 1.6±0.4 mmHg after infusion of 220±55 ml water and 4.0±1.0 mmHg after infusion of 273±69 ml nutrient drink (NS; figure 4A).

No significant changes were observed when comparing IGP during nutrient and viscous saline infusion (n=11). Maximum satiation was scored after 941±97 and 1088±112 ml nutrient and viscous saline infused respectively (NS). Maximum IGP decrease was 5.1±0.5 mmHg after 344±35 ml nutrient drink and 5.8±0.8 mmHg after 535±135 ml viscous saline respectively (NS; figure 4B).

7 volunteers were able to finish the solid meal in 11 minutes while 2 volunteers stopped eating at 9 and 10 minutes. Nevertheless, results from all volunteers were included in the analysis. In contrast with the nutrient drink the IGP did not decrease upon solid food intake, instead IGP increased continuously with a maximum IGP increase of 6.7±1.7 mmHg after 7.1±1.0 minutes, hereafter the IGP decreased so that the IGP at the end of the meal intake was 2.1±1.5 mmHg.

IGP during intragastric nutrient drink infusion: Effect L-NMMA

During intravenous infusion of L-NMMA, the baseline IGP significantly increased as compared to saline infusion (Figure 5A). Also during intragastric nutrient drink infusion the IGP was significantly increased during intravenous L-NMMA infusion. When compared to the average IGP in the 5 minutes preceding the intragastric infusion the maximal IGP decrease during intragastric nutrient drink infusion was 2.7±0.9 mmHg during L-NMMA infusion and 3.9±0.8 mmHg during saline infusion (NS).

Volunteers drank less during L-NMMA infusion (816±91 ml vs. 1032±71 ml; p<0.005).

Interestingly, when correlating the IGP increase from nadir with the corresponding satiation scores there was no significant difference between the correlation during saline or L-NMMA treatment: the correlation coefficients were 0.79±0.09 and 0.65±0.09 for saline and L-NMMA respectively, the linear regressions had a slope of 1.07±0.27 and 1.33±0.29 units satiation score increase per mmHg increase from nadir for saline and L-NMMA treatment, while the intercepts were 0.10±0.12 and 0.31±0.21 for saline and L-NMMA respectively (NS from 0).

FD patients vs. HVs

8 patients were selected and compared to 39 control experiments. Patients were satiated faster as compared to HV's (408±131 vs. 998±61 ml; p<0.0005). The maximum IGP decrease was 1.7±0.7 mmHg after infusion of 278±78 ml nutrient drink and significantly less pronounced as compared to the healthy control group (4.2±0.4 mmHg after 275±20 ml nutrient drink; p<0.01).

As can be seen in figure 6 were nadir IGP and/or ingested volume at maximum satiation in FD patients outside the normal range, represented by the 5% percentile as calculated from the healthy control data.

Discussion

During food intake the muscles of the proximal stomach are known to relax. This gastric accommodation reflex is known to provide a reservoir for the ingested food, while avoiding IGP increase (4; 16-18). The barostat is regarded as the golden standard to assess gastric accommodation: an intragastric bag is kept at constant pressure, while changes in bag volume are recorded to assess gastric muscle tone changes (19). However, the technique makes use of an inflated gastric balloon, which makes it particularly unphysiological to assess gastric accommodation during food intake (7;8). Moreover, its invasive nature excludes its application in routine clinical practice (9). Using a similar technique but controlling volume while measuring pressure, we showed that the IGP increase during inflation of an intragastric bag was significantly lower after ingestion of a nutrient drink as compared to ingestion of water. Since we previously showed, using a barostat, that ingestion of 200 ml nutrient drink evoked significant gastric relaxation (20), this experiment indicates that gastric accommodation lowers IGP of a distended stomach and that IGP during stomach distension can be used to assess gastric accommodation.

In order to setup a method to assess gastric accommodation by measuring the IGP during food intake we compared IGP while drinking nutrient drink or during intragastric infusion at different speeds. Independent on the speed or on the ingestion method IGP decreased initially to recover thereafter. Also during intragastric viscous saline infusion IGP decreased initially. No such pressure decrease was observed during water infusion, while inflating the intragastric bag or upon ingestion of solid food. Water very rapidly leaves the stomach (80% of a given volume empties in about 10 minutes ( 21)), hence we expect no or limited distension of the stomach during water infusion, especially since the infusion speed was only 15 ml min -1 . Increasing the viscosity and the osmolarity of a meal is known to delay gastric emptying (22;23); infusion of viscous saline therefore most likely distended the stomach also because the infusion speed was 60 ml min -1 . We postulate that the distension-induced relaxation of the stomach can only be seen using (semi)-liquid meals. Indeed, in contrast to solid meals, (semi)-liquid meals rapidly redistribute throughout the stomach (24), so that upon ingestion the different compartments of the empty stomach connect and that the IGP is equalized with low pressure zones in the fundus, effectively decreasing the pressure in the corpus region where our measurements took place. A solid meal on the other hand pools in the proximal stomach upon ingestion and does not immediately redistribute (25). We hypothesize that the solid meal accumulates in the proximal stomach around the manometer probe (similar to the intragastric balloon), leading to a higher pressure measurement. The IGP decrease upon fluid ingestion is likely dependent on the gastric muscle tone: with increased muscle tone (during L-NMMA treatment) the IGP decrease was indeed less pronounced, although this difference was not significant. An alternative explanation might be that activation of osmoreceptors in the duodenum induces gastric relaxation (26).

After the initial IGP decrease, IGP continuously increased upon further nutrient drink ingestion.

This IGP increase, which also occurred during solid food intake or during intragastric balloon distension, is probably a result of the increasing intragastric volume on one hand and the tone of the muscles of the stomach on the other hand. Indeed, after L-NMMA treatment IGP increase was more pronounced for a similar ingested volume. Interestingly, satiation ratings followed IGP increases in these experiments. Gastric distension has been shown to trigger stretch as well as tension mechanosensitive receptors that in turn relay their information via vagal and splanchnic nerves to the central nervous system (27;28). We hypothesize that increased IGP increases afferent signaling from the stomach, and could trigger increased feelings of satiation.

Most authors agree that (semi)-liquid meals are emptied from the stomach in a linear fashion with an emptying rate depending on the caloric content (rates vary from 0.4-2.5 kcal min -1 ) (29-33). In our study HV's consumed the nutrient drink at a rate of 22.5, 45 and 90 kcal min -1 . In a preliminary study using scintigraphy we examined gastric emptying of the nutrient drink used in the present study and found that after initial emptying of a small fraction, the meal further accumulates in the stomach throughout the test and that more than 80% remained intragastrically at the moment that maximum satiation is reached (34). We can therefore assume that gastric emptying plays a minor role in the determination of our results. However intestinal exposure of nutrients is a major determinant of satiation (35), and probably plays a role in the determination of satiation during our experiments, especially during low ingestion speed. Indeed, not only is gastric emptying relatively more important during low ingestion speed, the time of ingestion (and thus intestinal exposure of nutrients during ingestion) was considerably longer for low ingestion speed. This might explain why HV's consumed significantly more when ingestion speed was high and why the correlation coefficients for 15 ml min -1 were significantly smaller as compared to 30 and 60 ml min -1 , indicating that correlations became better with increasing ingestion speed. In light of the above-described findings we chose to proceed using an ingestion speed of 60 ml min -1 .

When comparing the IGP during drinking or intragastric infusion, no significant differences could be observed. However, since we were interested in the relationship between gastric accommodation and satiation we decided to continue with intragastric infusion experiments since results obtained while drinking might be more sensitive to factors that are not directly related to gastric accommodation such as taste (aversion), sensation of the nutrient drink while swallowing, and other subjective factors that might determine satiation or the volume consumed.

Several lines of evidence suggest that NO is a key mediator of gastric accommodation during food intake (36;37). We have previously shown in humans that inhibition of NO synthase with 8 ). Indeed, as described above the IGP during ingestion of solid and liquid meals is very different, and although gastric distension is an important satiation factor upon food intake, its role decreases in time as more and more satiation factors are released from the gut (35).

In all patients with unexplained meal-induced dyspeptic symptoms maximal volume ingested and/or IGP decrease were outside the normal range determined in HVs. This indicates that the present method can be used to objectively discriminate abnormal satiation and gastric accommodation to a meal.

In conclusion, IGP measurement during nutrient drink ingestion is a promising method to assess gastric accommodation and satiation during food intake in health and in patients with functional dyspeptic symptoms. Upon nutrient drink ingestion, IGP decreases initially to gradually recover thereafter. The IGP increase is correlated with satiation score increase and indicates that IGP is a determinant of satiation. We propose that IGP monitoring provides a minimally invasive, and more physiological alternative to the barostat for the assessment of gastric accommodation.

  that centrally involved mu-opioid receptors mediate an opposing effect.

FiguresFigure 1 :

 1 Figures

  Figure 1: (A) Pressure-volume curves obtained by gradually increasing isobaric gastric

Figure 2 :

 2 Figure 2: Mean (±S.E.M.) intragastric volume at 5-min intervals as measured by gastric barostat

Figure 3 :

 3 Figure 3: Intragastric pressure (IGP) change from baseline during intragastric nutrient drink

Introduction

  In between meals the proximal stomach has a high basal muscle tone mediated by the myoelectrical properties of the fundus (1) and by the constant cholinergic input mediated by the vagal nerves(2). Proximal gastric tone decreases upon food intake, a reflex relaxation that is mediated by different (para)sympathetic reflex pathways that have been shown to decrease the contractile cholinergic input and activate the release of nitric oxide (NO) (3). This gastric accommodation reflex increases the storage capacity of the stomach by increasing the compliance of the stomach muscles and thus keeps the intragastric pressure (IGP) low during food intake (4;5).

  mg kg -1 hour -1 L-NMMA impairs accommodation to a meal as assessed with a barostat(3). In the present study L-NMMA treatment increased IGP before and during nutrient drink infusion indicating that NO release mediates gastric tone in basal conditions and is indeed released during nutrient drink infusion. During L-NMMA treatment the HV's ingested significantly less nutrient drink. Interestingly, the increase in satiation per mmHg IGP increase was not different after L-NMMA treatment, indicating that IGP might be a determinant of satiation. Our findings are in contrast with a study by Vozzo et al. who found that treatment with 4 mg kg -1 hour -1 L-NMMA did not affect caloric intake or sensations of fullness or hunger(38). Despites the difference in the L-NMMA dose used we believe the dissimilar findings might be explained by the differences in food composition (solid meal in the study byVozzo et al.) and ingestion speed (in the study of Vozzo et al. volunteers had 30 minutes to eat as much as they could while in our study intragastric infusion lasted on average 14 and 17 minutes (during L-NMMA and placebo

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Representative example of a high resolution manometry recording ( participated in this example was on the left (lowest pressure indicated: mmHg (dark red)). The high-resolution that the most proximal sensors distal channels were positioned in the antrum (bottom of the figure). After a baseline recording nutrient drink was intragastrically inf interrupted line. Intragastric infusion (indicated by the second vertical interrupted line). Intragastric pressure was studied as the average pressure measured in esophageal sphincter (region indicated by an interrupted rectangular over the length of the recording).

Figure 4 :Figure 5 :Figure 6 :

 456 Figure 4: Intragastric pressure (IGP) change from baseline pressure during drink and water infusion (A; n=9), during nutrient drink and viscous saline infusion (B; n=11) and during nutrient drink infusion and while eating of a solid meal (C; n=9). mean ± S.E.M. until the average ingest volunteers were instructed to eat the meal in 11 minutes, 2 volunteers were unable to eat the whole meal and stopped at 9 and 10 minutes).

  Our aim was to setup and validate a technique to assess gastric accommodation by measuring the IGP during nutrient drink ingestion. Results obtained in healthy volunteers were compared with functional dyspeptic patients to assess the potential of this technique in the clinic.

	Methods
	Study subjects
	All study procedures were approved by the Ethics Committee of the Leuven University Hospital,
	Belgium. Written, informed consent was obtained from all subjects.
	Healthy volunteers (HVs): a total of 36 HVs (28 men, age: 28±1 years, body mass index: 22.1±0.4
	o F o
	r r
	P P
	e e r e e r
	R e v i e w R e v i e w

Alimentary Pharmacology & Therapeutic

Acknowledgements / Declaration of funding interests

Pieter Janssen is a postdoctoral research fellow of the FWO Flanders. This work was supported by an FWO grant and a Methusalem grant to Jan Tack, M.D., Ph.D.

Acknowledgements and disclosures

Pieter Janssen is a postdoctoral research fellow of the FWO Flanders. This work was supported by an FWO grant and a Methusalem grant to Jan Tack, M.D., Ph.D.

Pieter Janssen contributed to conception and design of the experiments, collection, analysis and interpretation of data and drafting the article. Sofie Verschueren and Huynh Giao Ly contributed to collection, analysis and interpretation of data. Jan Tack contributed to conception and design of the experiments, and revising the manuscript critically for important intellectual content

Summary 'box'

What is already known about this subject:

• Gastric accommodation keeps intragastric pressure (IGP) low during food intake

• The barostat, currently used to assess gastric accommodation, has important drawbacks that limit its use in the clinic

• Preliminary studies suggest that IGP measurement during food intake can be used to assess gastric accommodation What are the new findings:

• IGP decreases initially during nutrient drink ingestion and gradually increases thereafter The effect of nutrient drink on IGP was compared with tap water, a viscous saline solution and a solid meal. The viscous saline solution was custom-made so that the same osmolarity and viscosity as the nutrient drink was obtained (455 mOsm L -1 and 20 mPas sec -1 ) and contained 13.7 gr L -1 NaCl (Sodium Chloride ≥ 99% AR grade, Sigma-Aldrich, Bornem, Belgium) and 10 gr L -1 (hydroxypropyl)methyl cellulose (viscosity of 2% aqueous solution: 40-60 centipoises; Sigma-Aldrich, Bornem, Belgium), dissolved in tap water. The solid meal consisted of apple sauce, mashed potatoes, soft meat, and a pudding (4187 KJ, 46% fat, 36% carbohydrate, 18% protein (15)). The comparison between water and nutrient drink was performed using an infusion speed of 15 ml hour -1 , while the infusion speed of the experiment in which we compared the solid meal and the viscous saline solution with nutrient drink was 60 ml hour -1 .

For the solid meal the volunteers were instructed to eat the meal in 11 minutes so that they consumed at a similar energy rate as when they drank nutrient drink (378 KJ min -1 ).

Effect of N G -monomethyl-L-arginine (L-NMMA)

After introduction of the catheters and a 15-minute stabilization period, an intravenous infusion of either saline (100 ml hour -1 ) or L-NMMA (8 mg kg -1 hour -1 ) was started and continued for the remainder of the experiment. Allocation of the treatment was organized in a blind randomized cross-over fashion. We have used the same dose of L-NMMA in previous research in which we established a role for NO in gastric accommodation in man (3). Intragastric nutrient drink infusion started 20 minutes after the intravenous infusion.

Data analysis

Balloon distension

Per 50 ml volume increase intraballoon pressure was represented as an increase from baseline pressure (median pressure in the deflated balloon in the 2 minutes before inflation started), represented as mean ± S.E.M., and compared with a two-way repeated measures ANOVA followed by a Tukey post-test for multiple comparison (P<0.05 was considered significant).


Marzio ; Frazzoni
email: m.frazzoni@ausl.mo.it

Rita Conigliaro

Gianluigi Melotti

Fisiopatologia S Agostino

Digestiva Conigliaro

Rita ; Nuovo

Ospedale S Agostino

Gastroenterologia Melotti

Gianluigi Melotti

Fisiopatologia Digestiva -2 Gastroenterologia

Weakly acidic refluxes have a major role in the pathogenesis of PPI-resistant reflux oesophagitis

Keywords: GERD or GORD < Disease-based, Oesophagus < Organ-based, Acidity (intragastric) < Topics, Acidity (oesophageal) < Reflux oesophagitis -Acid reflux -Non-acid reflux -Proton pump inhibitors -

Weakly acidic refluxes have a major role in the pathogenesis of PPI-resistant reflux oesophagitis.

Introduction

Gastro-oesophageal reflux disease (GORD) has been defined as symptoms or mucosal damage produced by the abnormal reflux of gastric contents into the oesophagus [START_REF] Devault | Updated guidelines for the diagnosis and treatment of gastroesophageal reflux disease[END_REF]. Reflux oesophagitis, defined by the presence of visible mucosal breaks at conventional endoscopic examination of the oesophagus [START_REF] Lundell | Endoscopic assessment of oesophagitis: clinical and functional correlates and further validation of the Los Angeles classification[END_REF], is diagnosed in a minority of untreated GORD patients and is the most common consequence of oesophageal injury due to acid reflux (3) rather than the principal manifestation of GORD [START_REF] Vakil | The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus[END_REF]. Currently, the diagnosis of GORD relies on troublesome heartburn with/without regurgitation, endoscopic evaluation being reserved to patients with alarm symptoms or typical symptoms unresponsive to PPI therapy [START_REF] Vakil | The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus[END_REF][START_REF] Kahrilas | American Gastroenterological Association Institute Technical Review on the management of gastroesophageal reflux disease[END_REF].

Failure of proton pump inhibitor (PPI) therapy has become the most prevalent presentation of GORD in gastroenterology practice today. It is estimated that between 10% and 40% of patients with heartburn remain symptomatic on a standard dose of PPIs and many of them will continue to experience symptoms on even high doses of PPIs. Most of them originate from the group with nonerosive reflux disease (NERD) [START_REF] Fass | Management of heartburn not responding to proton pump inhibitors[END_REF].

Impedance-pH monitoring is a new diagnostic tool that can reliably detect acid and non-acid refluxes off and on PPI therapy. By means of this new diagnostic tool, reflux symptoms persisting despite acid suppressive therapy have been associated with either acid or non-acid gastroesophageal reflux [START_REF] Mainie | Acid and non-acid reflux in patients with persistent symptoms despite acid suppressive therapy: a multicentre study using combined ambulatory impedance-pH monitoring[END_REF][START_REF] Zerbib | Esophageal pH-impedance monitoring and symptom analysis in GERD: a study in patients off and on therapy[END_REF][START_REF] Frazzoni | Reflux parameters as modified by laparoscopic fundoplication in 40 patients with heartburn/regurgitation persisting despite PPI therapy. A study using impedance-pH monitoring[END_REF].

In patients with persisting typical reflux symptoms despite PPI therapy, upper endoscopy has been recommended (5) but reflux oesophagitis has rarely been found [START_REF] Ahlawat | A prospective study of gastric acid analysis and esophageal acid exposure in patients with gastroesophageal reflux refractory to medical therapy[END_REF][START_REF] Poh | Upper GI tract findings in patients with heartburn in whom proton pump inhibitor treatment failed versus those not receiving antireflux treatment[END_REF]. Thus far, patients with reflux oesophagitis detected despite ongoing PPI therapy have not yet been prospectively studied with impedance-pH monitoring. It is therefore not known if reflux oesophagitis detected despite ongoing PPI therapy is associated with abnormal acid or non-acid reflux.

In this study, we assessed acid and non-acid reflux parameters by means of impedance pHmonitoring in patients with persisting heartburn and with reflux oesophagitis detected despite ongoing PPI therapy. Our aim was to establish whether abnormal acid and/or non-acid reflux parameters are associated with PPI-resistant symptomatic reflux oesophagitis.

Materials and methods

Patients

We prospectively studied patients aged 18-80 years who underwent upper gastrointestinal endoscopy at our open-access service. Standard criteria were adopted to evaluate and report oesophageal abnormalities. Reflux oesophagitis was defined according to the Los Angeles Patients with heartburn persisting despite at least 8 weeks of continuous standard/high-dose PPI therapy and in whom reflux oesophagitis was found at endoscopic examination were asked to participate to the study. All patients received comprehensible and detailed explanations and signed a written informed consent. Patients with peptic structures and progressive systemic sclerosis were excluded. The study protocol was approved by our Institutional Review Board. All patients continued the PPI regimen that they were on at the time of entering the study, i.e. 30 min before breakfast or 30 min before breakfast and before dinner for the once daily and the twice daily dosage regimen, respectively.

Oesophageal manometry

Standard oesophageal manometry was performed before impedance-pH monitoring in all patients (MF). The station pull-through method was used to locate the lower oesophageal sphincter (LOS). Briefly, an 8-channel, water perfused manometry catheter was placed nasally into the stomach and slowly withdrawn. The lower oesophageal sphincter (LOS) was identified, and then, at each 0.5-cm station the resting pressure was measured at the end of expiration and the relaxation was assessed with wet swallows. Oesophageal body peristalsis was assessed and pressures measured with at least ten wet swallows. The upper oesophageal sphincter was finally identified and the catheter removed. All manometric data were processed using the Polygram Net module (Medtronic Functional Diagnostics A/S, Tonsbakken, Denmark) in order to obtain the mean LOS tone and the mean distal oesophageal amplitude (DOA).

Impedance-pH monitoring

Impedance-pH monitoring, always preceded by standard oesophageal manometry, was performed on PPI therapy (MF). An ambulatory, multi-channel, intra-luminal impedance system was used, consisting of a portable data logger and a combined impedance-pH catheter (Sleuth ambulatory system, Sandhill Scientific, Inc; Highland Ranch, CO). After LOS location by oesophageal manometry, the impedance-pH catheter was passed trans-nasally. The configuration of the catheter adopted allowed monitoring changes in intraluminal impedance at 3, 5, 7, 9, 15, and 17 cm above the LOS upper border. In addition, pH was monitored at 5 cm above and 10 cm below the LOS upper border. Subjects were discharged and were encouraged to maintain normal activities, sleep schedule, and eat their usual meals at their normal times. They were asked to remain upright during the day, and lie down only during their usual nocturnal bedtime. Event markers on the data-F o r P e e r R e v i e w 5 logger recorded symptoms, meal times, and posture changes. All studies were performed for 24 h, after which patients returned to the lab for catheter removal and data review. Impedance, pH, and symptom signals were collected at a resolution of a 50-Hz sampling rate on a 256-MB compact flash card for later download. Impedance and pH information was analysed using a dedicated software program (BioView Analysis, Sandhill Scientific, Inc; Highland Ranch, CO) and visual analysis (MF). Analysis included identification, enumeration and characterization of individual reflux events and their relationship with symptoms. Meal times were excluded. Liquid-only reflux was defined as a retrograde 50% fall in impedance from baseline in the two distal impedance sites.

Gas reflux was defined as a rapid increase in impedance >3,000 ohms, occurring simultaneously at least in two oesophageal measuring segments. Liquid-gas (mixed) reflux was defined as gas reflux occurring immediately before or during a liquid reflux. Gas reflux events without liquid (belches) were considered separately and not included in the analysis. The time period with gastric pH < 4, i.e. the percentage gastric acid exposure time (GAET), was computed for the total study period as well as the time period with oesophageal pH < 4, i.e. the percentage oesophageal acid exposure time (OAET). Using the pH tracings, reflux events were classified as: i) acid (nadir pH<4), ii) weakly acidic (nadir pH between 4 and 7) or iii) weakly alkaline (nadir pH not below 7) refluxes [START_REF] Sifrim | Gastro-oesophageal reflux monitoring: review and consensus report on detection and definitions of acid, non-acid, and gas reflux[END_REF]. Data analysis was performed on liquid and mixed reflux episodes for acid, weakly acidic and weakly alkaline refluxes in the distal oesophagus. The number of proximal reflux events (reaching the 15cm impedance site) was also assessed. The 95 th percentile of values obtained in 20 healthy controls studied in the same way at our centre (13) served as the upper normal limit.

Statistics

Reflux parameters were expressed as the median and interquartile range. Correlation between GAET and OAET was assessed using the Spearman rank correlation test. To analyse the differences between patients on once daily and twice daily PPI regimens, the Mann-Whitney test and the Fisher's exact test were used as appropriate. A P <0.05 was considered significant.

Results

Between March 2007 and September 2010, 20 patients with persisting heartburn and reflux oesophagitis detected despite ongoing PPI therapy of at least 8-week duration were studied. Their baseline characteristics are reported in Table 1; the LOS tone was lower than 10 mmHg in 7 (35%) cases. The median GAET was 38% (range 10%-81%); it was >30% in 14 (70%) patients. There was no significant correlation between the GAET and the OAET (r s = 0.072, P NS).

The median and interquartile range of the main reflux parameters are reported in Table 2.

The OAET was higher than normal in 4 (20%) patients; the number of acid, weakly acidic and weakly alkaline refluxes was above the normal range in 5 (25%), 20 (100%) and 3 (15%) patients, respectively (Figure 1).

Ten patients were on a once daily regimen and ten patients were on a twice daily regimen of their particular PPI. The GAET was >30% in eight (40%) patients on a once daily regimen and in six (30%) patients on a twice daily regimen (P NS). Median values of GAET and reflux parameters did not differ significantly between the two subgroups of patients (Table 3).

Discussion

In this study, we prospectively assessed acid and non-acid reflux parameters in patients with persisting heartburn and with reflux oesophagitis detected despite ongoing assumption of a standard/high-dose PPI therapy from at least 8 weeks. We found abnormal acid and weakly alkaline reflux parameters in a small minority of patients whereas the number of weakly acidic refluxes was above the normal range in all cases.

Healing of mucosal lesions in reflux oesophagitis represents a reliable end point for success of therapy and correlates well with improvement of heartburn (4). Ninety percent healing rates have been reported with standard PPI dosages after 8 weeks of treatment [START_REF] Castell | Esomeprazole (40 mg) compared with lansoprazole (30 mg) in the treatment of erosive esophagitis[END_REF]. Accordingly, in a recent study reflux oesophagitis has been detected in less than 7% of patients with persisting heartburn despite PPI treatment [START_REF] Poh | Upper GI tract findings in patients with heartburn in whom proton pump inhibitor treatment failed versus those not receiving antireflux treatment[END_REF], a result confirming that the vast majority of PPI-unresponsive GORD patients belong to the NERD group [START_REF] Fass | Management of heartburn not responding to proton pump inhibitors[END_REF].

Healing of reflux oesophagitis has been positively related to the percentage of time intraoesophageal [START_REF] Holloway | Relation between oesophageal acid exposure and healing of oesophagitis with omeprazole in patients with severe reflux oesophagitis[END_REF] and intra-gastric pH ( 16) are >4. Recently, maximal reflux oesophagitis healing rates have been related to an intra-gastric pH >4 for 70% or more of the 24-h period: intra-gastric pH control has then been proposed as a clinically relevant measurement of drug efficacy in the treatment of patients with moderate to severe reflux oesophagitis [START_REF] Katz | A model of healing of Los Angeles grades C and D reflux oesophagitis: is there an optimal time of acid suppression for maximal healing?[END_REF]. In our patients, one half of whom treated with a standard PPI dosage, an intra-gastric pH >4 was achieved for 70% of the time in only 30% of cases as a GAET >30% was detected in 70% of cases. However, the OAET and the number of acid refluxes were above the normal range only in 20% and 25% of cases, respectively: these findings are to be related to the lack of correlation between GAET and OAET (r s = 0.072, P F o r P e e r R e v i e w 7 NS), in accordance with previous studies [START_REF] Frazzoni | Reflux patterns in patients with short segment Barrett's oesophagus: a study using impedance-pH monitoring off and on proton pump inhibitor therapy[END_REF][START_REF] Roman | Correlation between gastric pH and gastro-oesophageal reflux contents: ambulatory pH-impedance monitoring results[END_REF], and confirm the multi-factorial pathogenesis of GORD.

As we found a higher than normal number of weakly acidic refluxes in all our patients, we believe that weakly acidic refluxes have a relevant role in the pathogenesis of PPI-resistant reflux oesophagitis. Reflux oesophagitis was originally termed "peptic" [START_REF] Winkelstein | Peptic esophagitis (a new clinical entity)[END_REF], i.e. an oesophagitis resulting from the irritant action on the mucosa of hydrochloric acid and pepsins. Indeed, the presence of pepsins is critical for oesophageal mucosal damage to occur: activation of these proteolytic enzymes occurs at pH < 4.5, proteolytic activity is maintained up to pH 5.5 and denaturation occurs at pH > 7 [START_REF] Roberts | Review article: human pepsins -their multiplicity, function and role in reflux disease[END_REF]. We considered separately weakly acidic refluxes from weakly alkaline refluxes as peptic mucosal damage would not be expected when reflux occurs at a pH > 7. Weakly alkaline refluxes were above the normal range in a small minority (15%) of cases only. Therefore, we believe that weakly alkaline refluxes have a little role, if any, in the pathogenesis of reflux oesophagitis. On the other hand, as the lowest GAET value that we detected was 10% and as in 70% of our patients the GAET was >30%, activation of pepsins may have well occurred in the stomach of all our patients, both those on once daily as well as those on twice daily PPI therapy. Therefore, activated pepsins may have been present in a relevant proportion of weakly acidic refluxes detected in our patients, proteolytic activity being maintained when the pH was <5.5. As we found a higher than normal number of weakly acidic refluxes in all our patients, presence of activated pepsins in a weakly acidic oesophageal environment during reflux episodes may represent the main pathogenic mechanism by which visible oesophageal mucosa damage may be found in some GORD patients despite ongoing PPI therapy. Our results confirm that weakly acidic reflux is one of the major underlying mechanisms of refractory GORD [START_REF] Tsoukali | The role of weakly acidic reflux in proton pump inhibitor failure, has dust settled?[END_REF].

Despite one half of our patients were on a once daily PPI regimen, we found abnormal acid reflux parameters in a minority of them and independently of the PPI dosage schedule. Therefore, we agree that treating patients on the basis of impedance-pH monitoring results rather than treating them empirically will result in increased treatment success [START_REF] Becker | Clinical trial: persistent gastro-oesophageal reflux symptoms despite standard therapy with proton pump inhibitors -a follow-up study of intraluminal-impedance guided therapy[END_REF]. However, impedance-pH monitoring should include oesophageal as well as gastric pH monitoring, as the efficacy of incremental PPI dosages for those patients (30% of cases in our series) with a GAET <30% would probably be low [START_REF] Katz | A model of healing of Los Angeles grades C and D reflux oesophagitis: is there an optimal time of acid suppression for maximal healing?[END_REF]. Recently, we have shown that laparoscopic fundoplication improves significantly symptoms as well as acid and weakly acidic reflux parameters when compared with high-dose PPI therapy in refractory GORD patients [START_REF] Frazzoni | Reflux parameters as modified by laparoscopic fundoplication in 40 patients with heartburn/regurgitation persisting despite PPI therapy. A study using impedance-pH monitoring[END_REF]. Our findings support the view that therapeutic interventions in PPI-resistant GORD with/without reflux oesophagitis should be guided by impedance-pH monitoring, in order to identify those patients who would benefit more from laparoscopic fundoplication than from incremental PPI dosages [START_REF] Spechler | Surgery for gastroesophageal reflux disease: esophageal impedance to progress?[END_REF]. The vast majority of our patients were males, as expected in patients with reflux oesophagitis [START_REF] Frazzoni | Pathophysiological characteristics of the various forms of gastro-oesophageal reflux disease. Spectrum disease or distinct phenotypic presentations?[END_REF], and had a hiatal hernia which adversely affects the efficacy of PPI therapy [START_REF] Frazzoni | Hiatal hernia is the key factor determining the lansoprazole dosage required for effective intra-oesophageal acid suppression[END_REF].

As our patients had active reflux oesophagitis, basal impedance was sometimes low in our series, but we confirm that this does not preclude the recognition of reflux episodes during visual analysis [START_REF] Sifrim | Gastro-oesophageal reflux monitoring: review and consensus report on detection and definitions of acid, non-acid, and gas reflux[END_REF][START_REF] Frazzoni | Reflux patterns in patients with short segment Barrett's oesophagus: a study using impedance-pH monitoring off and on proton pump inhibitor therapy[END_REF]. Our upper normal limits, defined by the 95 th percentile of values obtained in 20 healthy controls at our centre [START_REF] Frazzoni | Reflux patterns in patients with short segment Barrett's oesophagus: a study using impedance-pH monitoring off and on proton pump inhibitor therapy[END_REF] are at variance with those reported by previous series [START_REF] Shay | Twenty-four hour ambulatory simultaneous impedance and pH-monitoring: a multicenter report of normal values from 60 healthy volunteers[END_REF][START_REF] Zerbib | Normal values and day-to-day variability of 24-h ambulatory oesophageal impedance-pH monitoring in a Belgian-French cohort of healthy subjects[END_REF]. This can be explained by different dietary habits: our normal values were obtained in normal subjects eating their usual meals in a Mediterranean region and are very close to those found in our country when a standardized Mediterranean diet was adopted [START_REF] Zentilin | Normal values of 24-h ambulatory intraluminal impedance combined with pH-metry in subjects eating a Mediterranean diet[END_REF]. Moreover, there is evidence that the OAET is lower when impedance-pH instead of pH-only criteria are adopted to detect acid reflux [START_REF] Hila | Combined multichannel intraluminal impedance and pH esophageal testing compared to pH alone for diagnosing both acid and weakly acidic gastroesophageal reflux[END_REF].

In conclusion, we have found that in patients with persisting heartburn and with reflux oesophagitis detected despite ongoing PPI therapy, weakly acidic refluxes were above the normal range in all cases whereas acid reflux parameters and weakly alkaline refluxes were normal in the vast majority of cases. We have shown that gastric acid secretion, with consequent intra-gastric pepsins activation, may persist despite ongoing PPI therapy: activated pepsins may well be present in a relevant proportion of weakly acidic refluxes. As activated pepsins maintain their proteolytic activity in a weakly acidic environment, they may be responsible for mucosal damage. We conclude that weakly acidic refluxes have a major role in the pathogenesis of PPI-resistant reflux oesophagitis. Therapeutic interventions in patients with PPI-resistant reflux oesophagitis should be tailored on the basis of impedance pH-monitoring results.

F

  . Hiatal hernia was defined as a distance between the diaphragmatic hiatus and the gastroesophageal junction >2 cm.

14 Table 3 .

 143 Figure 1

Figure 1 .

 1 Figure 1. Abnormal and normal reflux parameters in 20 patients with symptomatic reflux oesophagitis detected on PPI therapy

Table 1 .

 1 Baseline characteristic of 20 patients with symptomatic reflux oesophagitis detected on PPI therapy

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 Twenty-four-hour impedance-pH monitoring values in 20 patients with symptomatic reflux oesophagitis detected on PPI therapy

	Median	Upper normal limit
	(interquartile range)

Alimentary Pharmacology & Therapeutic

Financial support

The study was conducted without any financial support.

Statement of interests

There are no affiliations that might lead to bias or a conflict of interest.