BACKGROUND:

Dilation of intercellular spaces (DIS) of human esophageal epithelium, evident at transmission electron microscopy (TEM), is an early marker of damage caused by gastroesophageal reflux, but its reversibility after therapy has not been investigated.

AIM:

To evaluate whether omeprazole can induce the healing of DIS.

METHODS:

Thirty-eight symptomatic patients, 22 with nonerosive reflux disease (NERD) and 16 with erosive esophagitis (EE), classified on the basis of 24-h pH monitoring, were enrolled. During upper gastrointestinal endoscopy, six biopsies from apparently normal mucosa were taken within the lower 5 cm of the esophagus for histological and TEM analysis. One hundred computer measurements were taken on TEM photomicrographs of the specimens in each patient. After 3 months of omeprazole 40 mg/die a further endoscopy with biopsies was performed. In patients with persistent heartburn and/or incomplete ultrastructural recovery of esophageal epithelium, a new endoscopy was performed after 3 more months of treatment.

RESULTS:

After 3 months of therapy, 35 patients (92.1%) showed a complete recovery of DIS and resolution of heartburn. Three patients required 3 more months of therapy because of an incomplete recovery of the epithelium correlated with sporadic heartburn. Healing of the mucosa was achieved in two patients, whereas one had an incomplete recovery of DIS with persistent heartburn.

CONCLUSIONS:

Three and six months of omeprazole therapy led to a complete recovery of DIS in 92.1% and 97.4% of cases, respectively. No significant differences of DIS between NERD and EE were noted. Complete recovery of DIS was accompanied by regression of heartburn in all cases.

INTRODUCTION

Gastroesophageal reflux disease (GERD) is a common condition affecting up to 30% of adults (¹). The severity and frequency of GERD symptoms, typically heartburn and acid regurgitation, are not predictive of the presence of esophageal lesions at endoscopy (²) and almost 70% of symptomatic subjects do not demonstrate erosions (³).

Electron microscopy recently disclosed that symptomatic patients with positive 24-h pH monitoring, with or without erosive esophagitis, have ultrastructural signs of esophageal damage, i.e., dilation of intercellular spaces (DIS). In addition, this marker showed that symptomatic patients with or without erosions have a similar ultrastructural rate of esophageal mucosa damage in apparent undamaged epithelium. (⁴).

Meta-analysis of acid suppression induced by proton pump inhibitors (PPIs) show that the healing proportion of erosions is directly related to the degree and duration of acid suppression (^5,6,7,8). Nevertheless, there is currently no evidence of a relation between regression of symptoms and effective healing of esophageal ultrastructural damage in nonerosive esophagitis (NERD).

The aim of this study was to establish whether omeprazole treatment can induce an ultrastructural healing of the esophageal epithelium both in erosive and nonerosive esophagitis in addition to a regression of symptoms.

MATERIAL AND METHODS

Patients gave written informed consent to participate in the study which was approved by the local research ethical committee in June 2001. The design of the study was single blinded for the histological and ultrastructural evaluation.

Study Populations

Thirty-eight patients (18 men) had GERD defined by typical symptoms (recurrent episodes of heartburn and/or acid regurgitation) and abnormal 24-h pH parameters. Exclusion criteria were: patients with Barrett's esophagus, asymptomatic patients with positive pH-metry or patients undergoing NSAID or steroid therapy, subjects with peptic ulcerations, pyloric stenosis, gastric resection, or other severe organ diseases.

General Study Design

All patients stopped taking medications known to affect gastrointestinal motility or acid secretion at least 2 wk before the beginning of the study. After calibration, a pH electrode was passed nasally and positioned 5 cm above of the lower esophageal sphincter. Subjects were sent home with instructions to keep a diary of symptoms, meal times, time of going to bed, and getting up. Subjects returned the next day after 22–25 h of monitoring to have the probes removed and the diary reviewed. Within 7 days, an upper gastrointestinal endoscopy was performed and six biopsy specimens were taken from each patient. The biopsies were obtained from the lower 5 cm of esophagus from normal appearing mucosa for histological and ultrastructural evaluation (three biopsies, respectively). After the initial assessment of ultrastructural damage, all patients were treated with omeprazole 40 mg once daily, 30 min before breakfast, for 3 months keeping a diary of symptoms (heartburn and acid regurgitation). The frequency and severity of symptoms were graded as reported in Table 1. GERD was defined as the presence of at least weekly symptoms of heartburn (⁹). At the end of this period a further upper endoscopy with six esophageal biopsies was performed.

Table 1 - Graduation of frequency and severity of symptoms (heartburn an/or acid regurgitation).

Full table

Patients with persistent heartburn and/or an incomplete recovery of esophageal epithelium at TEM underwent a further endoscopy after 3 further months of treatment with omeprazole 40 mg daily.

Ambulatory Esophageal pH Monitoring

A prolonged esophageal study was performed using an antimony electrode. The pH electrode (Synectics Med., Stockholm, Sweden) was calibrated at 37°C in pH 7.01 and pH 1.07 buffer solution (Fisher Scientific, Fairlawn, NJ) before and after completing each study. An Ag/Cl reference electrode was placed on the anterior chest. The pH electrode was connected to a portable digital data recorder (Flexilog 2000, Oakfield Instruments Ltd., Witney, U.K.) that stored pH data every 4 s for up to 24 h. At the end of the study, information in the data recorders was downloaded into a PC for analysis using a Flexisoft III computer program (Oakfield Instruments Ltd., U.K.). According to Johnson's methodology (¹⁰), gastroesophageal reflux was considered in every drop of intraesophageal pH to be less than 4.0 for at least 20 s.

Endoscopic Evaluation

All subjects underwent upper GI endoscopy (videogastroscope Olympus GIF V2, Hamburg, Germany) after sedation by IV administration of midazolam (2.5 mg). Six biopsies were obtained from the lower 5 cm of esophagus from areas of macroscopically noneroded esophageal mucosa. The presence of esophagitis was noted and graded according to the Los Angeles classification (¹¹).

Histologic Evaluation

Three specimens from each patient were fixed in formalin immediately after endoscopy and then embedded in paraffin wax. Serial sections of 4 m thickness were cut and stained with hematoxylin–eosin. Esophagitis was identified and graded according to the Ismail–Beigi classification (^12,13).

Transmission Electron Microscopy (TEM)

Three specimens from each patient were fixed in glutaraldehyde, rinsed, and processed for TEM. The specimens were postfixed in 1% buffered osmium tetroxide. They were then dehydrated through a graded alcohol series and embedded in Araldite. Blocks were trimmed and ultra-thin sections on copper grids were post-stained with uranyl acetate and lead citrate. Each specimen was analyzed by TEM (Philips 410, Eindhoven, Netherlands) and then photographed at an accelerating voltage of 80 kV. Photographs of at least of 10 significant fields were magnified to 3500.

Morphometric Analysis

Ten TEM photomicrographs of biopsy specimens from each patient were obtained. In particular, the suprabasal layer of the esophageal mucosa was examined in each image. Photographs with an internal scale marker were digitized and then each field was evaluated using the EndoxPro System (Casti imaging, Medra-Venice, Italy). At least 10 randomly selected perpendicular transects to adjacent membranes were drawn and measured in each image for a total of 100 measurements in each case. Every transect was drawn at a distance not closer than 1 m. A mean score of DIS of 0.74 m was considered a cutoff score for damage (⁴).

Statistics

Measurements obtained were used to calculate mean DIS scores and mean scores of maximum DIS for each subject and for all cases as a whole. Statistical significance was determined using Student's t-tests for paired and unpaired samples. The results of treatment were compared by ² test for comparison of proportion with a 95% confidence interval (CI). All statistical analyses were 2-tailed, and significance was accepted at a p value < 0.05. Data were analyzed with a ©SPSS Inc. software.

RESULTS

Thirty-eight patients (mean age 41.08 12.48 yr, range 25–70 yr) had GERD as defined by typical symptoms (recurrent episodes of heartburn or acid regurgitation) and abnormal 24-h pH parameters. Of these, 22 had a normal appearance of esophageal mucosa at endoscopy (NERD) (mean age 40.3 10.76 yr, 10 men), whereas 16 had EE (mean age 43.69 13.88 yr, 8 men). At histology, of the 16 patients with EE, 8 had mild esophagitis, 1 had moderate esophagitis and 7 had normal histological pattern. No patients with NERD showed histological signs of esophagitis (Table 2).

Table 2 - Demographic data and macroscopic and histological features at baseline.

Full table

At TEM dilation of intercellular spaces >0.74 m (mean value of 100 intercellular space diameters) was used as a marker of esophageal damage and all symptomatic patients presented this pattern.

After 3 months of therapy 35/38 patients (92.1%, CI 84.9–97.3%, 2 = 26.95 p 0.0001) showed a complete recovery of the esophageal epithelium at TEM (Fig. 1) and a disappearance of symptoms. Among them, 15/16 patients (93.75%, CI 84.5–99.8%) were affected by EE and 20/22 patients (90.9%, CI 81.3–99.6%) by NERD (Table 3).

Figure 1.

Photomicrographs of oesophageal mucosa, obtained using transmission electron microscopy (TEM) of the suprabasal layer (original magnification, 3500). A and C: patients with NERD and erosive oesophagitis, respectively, before treatment. B and D shows resolution of DIS after treatment.

Full figure and legend (368K)

Table 3 - Mean, mean of maximum DIS score, and maximum DIS score evaluated from TEM images of biopsy samples from patients with NERD or EE at baseline and after 3 months of treatment.

Full table

Three patients (mean age 38.06 6.4yr, range 32–44 yr), one with NERD and two with EE, required 3 further months of therapy because of an incomplete healing of the mucosa and persistence of symptoms.

After this period, a complete recovery of esophageal mucosa and heartburn was achieved in two patients (one with NERD and one with EE) (Fig. 2) so that 37 of 38 patients (97.37%, CI 88.4–99.7, 2 = 34.11 p 0.001) presented a complete recovery of DIS and resolution of symptoms. In one patient with EE we observed a reduction of DIS and the persistence of sporadic and moderate heartburn (Table 4).

Figure 2.

Effect of omeprazole therapy on the reduction of intercellular spaces (ICS) of oesophageal mucosa in patients with NERD or erosive oesophagitis. The dashed line represents the mean score of DIS which is the cut off (0.74 m) for damage.

Full figure and legend (22K)

Table 4 - Mean value of DIS and Grading of Symptoms in Three Patients With an Incomplete Healing of Mucosa after 3 Months of Therapy and Outcome after 3 Further Months of Treatment.

Full table

DISCUSSION

Among the antisecretive drugs, PPIs offer rapid symptomatic relief in the highest percentage of patients with GERD due to their inhibition of gastric acid secretion with suppression of distal esophageal acid exposure (^14,15); it has not demonstrated a direct influence of PPI on esophageal mucosa, to date. A recent meta-analysis of speed of healing and symptoms relief in GERD showed that PPIs heal EE in highest proportion of patients when compared with all other drug classes (¹⁶). Moreover, PPIs are effective in healing the symptoms of NERD (¹⁷), a major clinical problem requiring a refocus of attention and a priority for clinical research (¹⁸). Carlsson et al. (¹⁹) have confirmed the efficacy of PPI evaluating the effect of this therapy on esophageal mucosa integrity using the assessment of transmural electric potential difference. In fact, patients treated with omeprazole, when it is instilled HCl in the esophagus, present an improved pattern of the epithelial resistance to the acidic insult with a lower diffusion potential.

The clinical improvement after PPI treatment in patients with EE can be related to the endoscopic disappearance of EE; while in the case of NERD, where the esophageal mucosa appears normal at endoscopy and frequently also at histology, there is not a parameter that can explain the improvement of symptoms after treatment (²⁰).

Recently, TEM demonstrated that the DIS of esophageal epithelium is a highly sensitive and early marker of damage induced by reflux into the esophagus. This marker is present in both EE and in NERD where it represents the only reliable parameter of damage found to date (⁴).

We analyzed at TEM several esophageal biopsies taken during endoscopy performed on 38 subjects treated with omeprazole 40 mg daily for 3 months, irrespective of the presence of EE or normal appearing mucosa, but with abnormal esophageal acid exposure at pH-metry. After this period, a new endoscopy was performed and symptoms were investigated.

At first, 92% of patients presented a complete resolution of symptoms whereas three patients required 3 more months of therapy because of the persistence of symptoms. At the end of this period, one patient showed a partial regression of symptoms. At endoscopy, our findings on the efficacy of omeprazole treatment on EE agreed with previous studies (^21,22,23,24): a macroscopic recovery of EE was achieved after 3 months of therapy in all patients.

At TEM we observed a complete recovery of DIS in 92.1% and in 97.4% after 3 and 6 months of therapy, respectively. No significant differences between NERD and EE were seen. The ultrastructural healing of the esophageal mucosa was accompanied by a complete resolution of the esophageal symptoms in all cases. The three patients with persistent symptoms after 3 months of therapy, showed an incomplete healing of DIS. Two of them, who became asymptomatic after a further 3 months of therapy, showed a complete ultrastructural recovery of the mucosa. The one patient who had still an incomplete resolution of symptoms after 6 months of treatment showed only a partial regression of DIS at TEM. The correlation between symptoms of relief and ultrastructural healing of the mucosa suggests a link between the integrity of intercellular spaces and stimulation of sensory intraepithelial neurons (²⁵).

In conclusion, this is the first demonstration that omeprazole may induce, in our subset of patients, the ultrastructural healing of mucosal damage both in EE and in NERD and in asymptomatic subjects after treatment there is a complete recovery of DIS. We do not know whether this event affects the natural history of the disease. DIS is a reliable marker of damage and should be proposed as a healing parameter for controlled clinical trials in NERD patients. Additional studies are required to assess the ultrastructural esophageal mucosa in patients with NERD, without pathological pH-metry, and in patients with duodenogastroesophageal reflux. Moreover, the intrinsic capacity of defense of the epithelium from damaging factors may be a key to disclose one of the unknown potential causes of inappropriate answer to PPI therapy.

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Acknowledgements

The authors thank Mr Paolo Borsetti, Walter Mantovani, and Anne Prudence Collins for excellent technical assistance.