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Glucose infusions into stomach or jeiunum decrease gastric injury from restraint stress
GASTROENTEROLOGY Vol. 114, No. 4 A l l 4 AGA ABSTRACTS
• G0468 A NOVEL
REAL-TIME QUANTITATIVE PCR ASSAY FOR DETECTION OF HELICOBACTER PYLORI IN THE GASTRIC MUCOSA. M. Endoh, T. Shimada, N. Watanabe, Y. Ohtsuka, K. Sakata, T. Sasai, H. Hiraishi, A. Terano. Second Department of Internal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0207, Japan. BACKGROUND: Although the assessment of the density of Helicobacter pylori (H. pylori) colonization may be important, precise quantitative evaluation of H. pylori in the gastric mucosa has been difficult. We have developed a novel real-time quantitative PCR technique based on 5' -3' exonuclease assay for detection of H. pylori. Using this system, we evaluated number of H. pylori organisms present in gastric biopsy specimens. METHODS: A primer pair for PCR amplification was made according to the reported sequence of ureA gene of H. pylori, and an oligonucleotide probe labeled with two fluorescent dyes (a reporter dye at the 5' end and a quencher dye at the 3' end) was also designed. While the probe is intact, the quencher dye greatly reduces the fluorescent signal by the reporter dye (Fluorescence resonance energy transfer phenomenon). During the PCR extension phase, the annealed probe is cleaved by 5' -3' exonuclease activity of Taq DNA polymerase, increasing the reporter dye signal. The reporter dye signal during a PCR assay was monitored with a ABI PRISM 7700 Sequence Detector (Perkin-Elmer). Purified PCR products of ureA gene were used as standard samples for making standard curves plotting log starting copy number and threshold cycle (Ct: PCR cycle number at which the reporter fluorescence signal reached above baseline). Biopsy specimens were collected from antral mucosa of consecutive 32 patients underwent gastroscopy (GU 9, DU 9, GC 1, NUD 13). Three biopsy specimens were obtained from each patient to avoid the effect of patchy distribution of H. pylori. DNA was extracted by conventional phenol-chloroform method. Since the size of each biopsy specimen was variable, quantitative PCR assay described above was performed using 0.5 ~tg of DNA extracted from each sample. !~_!LL_.T~: In each amplification reaction, a liner standard curve could be generated in a wide range of starting copy numbers (6xl07 to 6xl0 copies of the target sequences, R2 > 0.99). Nine of 32 samples (DU 2, NUD 7) showed no increase in the fluorescence signal during PCR process, indicating the absence of H. pylori, which was also confirmed by histological examinations. In H. pylori positive patients, the detected ureA copy numbers (i.e. number of H. pylori organisms) were variable among patients (range 1.4x102 - 1.2x105 copies/0.5Mg DNA, mean 2.6x104 copies/0.51ag DNA). The average ureA copy numbers of samples from DU and GU patients were not significantly different from that of NUD patients [GU (2.1 _+ l.l)xl04, DU (1.8 _+0.6)xl04, NUD (2.6 + 1.1)xl04 copies/0.51ag DNA, respectively]. The sample of one GC patient showed high density of H. pylori colonization (1.2x105 copies/0.5Mg DNA). CONCLUSIONS: Accurate quantitation of H. pylori in biopsy specimens was possible with this PCR system, and it will be useful for 1-1.pylori research. G0469
GLUCOSE INFUSIONS INTO STOMACH OR JEJUNUM DECREASE GASTRIC INJURY FROM RESTRAINT STRESS. K. Ephgrave, A. Ong, K. Broadhurst, J. Cullen. VAMC and University of Iowa Hospital College of Medicine, Iowa City, Iowa. Intragastric 25% glucose (D25) at 2ml/hr during restraint stress prevents mucosal injury, increases intragastric pH, and suppresses stress-induced hypercontractility in rats. Intrajejunal glucose was equally protective at this rate, but with glucose reflux into the stomach. The purpose of the present study was to test the hypothesis that enteral glucose can initiate gastric protection without contacting gastric afferents. Methods: Male 200-300 gm Sprague-Dawley rats were prepared with gastric, jejunal, and jugular catheters, and a strain gauge transducer on the anterior surface of the gastric antrum. After 5 days recovery, they underwent a restraint protocol at room temperature for 2 hours followed by cold (4 °) for an additional 2 hours, while receiving .5 ml/hr saline IG (n=6), D25 IG (n=6), D25 IJ (n=6), or saline IJ (n=3). Gastric lesion length, volume, pH, glucose, and mucin were measured following stress. Gastric motility was measured continuously and blood glucose was measured terminally. Findings: Lesion length (mm) decreased in both of the groups receiving D25, (IG saline 10.1 -+ 1.8, IG D25 4.0 _+ 1.4", IJ D25 2.5 _+ 1.0", IJ saline 5.7 _+ 1.4, *p < .01 vs. saline). No effect was seen on gastric pH (IG saline 2.2_+.2, IG D25 2.1 _+.3, IJ D25 2.1 _+.2, IJ saline 1.9 _+.1), nor mucus (29.7 _+4 vs. 25.1 _+9 U, IJ D25 vs. IJ saline). Stressinduced gastric hypercontractility regressed for < 10 minutes following IG D25, but then recurred. No hyperglycemia occurred, and no jejunal glucose refiuxed intragastrically, as gastric levels were 611 + 28 mg/dl for IG D25 versus 67 + 11 forlJ D25, p < .01. Conclusions: Small volumes of jejunal or gastric glucose ameliorate restraintinduced gastric injury, though jejunal infusion does not affect gastric glucose levels. The infusions did not affect gastric contractions, acidity, or mucin production, so the protective mechanism is unknown. Study supported by VA Merit Review Funds
G0470
EFFECT OF ERADICATION ON DYSPEPTIC SYMPTOMS OF HELICOBACTER PYLORIoPOSITIVE PATIENTS. H.J. Epple 1, C. Bojarski 1,2, F.W. Kirstein 1, M. Fromm2, E.O. Riecken l, J.D. SchulzkeL Depts. of ]Gastroenterology & Infectiology and 2Clinical Physiology, UKBF, Freie Universit~it Berlin, D- 12200 Berlin, Germany The subgroup of patients with dyspepsia for whom eradication therapy can be recommended has not been defined so far. In this study, we assessed the effect of Helicobacter pylori (HP) eradication on dyspeptic symptoms of a highly selected group of HP positive patients. re*0:
7o.
m,
First treatment
~zo~~a°~ E successf(n=l ul O)
unlriel thempy (n=7)
pemieten4H P-infecti~
10
before'therapy
altethrerapy
therapy
therapy
retherapy
Inclusion criteria were dyspepsia for at least 6 months, HP infection (diagnosed by laC-urea breath test (UBT) and histology), no active or previous peptic ulcer. After gastroscopy the patients underwent abdominal sonography, H2-exhalation test with lactose, and 24 h oesophageal pHmonitoring in order to exclude organic diseases causing dyspepsia. The remaining patients received eradication therapy. Eradication was confirmed by 13C-UBT 6 weeks after completion of the therapy. The severity of dyspeptic symptoms was monitored with a validated score in a blinded fashion before and 6 months after therapy or retherapy, respectively. From an initial number of 128 patients 87 patients were excluded after endoscopy and the other investigations. 24 patients refused additional investigations after the endoscopy. 17 patients without other organic disease except for HP gastritis were treated with eradication therapy. In 10 patients the first treatment was successful, while 7 patients were HP-negative only after retberapy. The symptoms of successfully treated patients improved considerably (see Figs.), whereas symptoms of patients with persistent infection remained unchanged (AS15 vs A0, p < 0.05) and only improved after successful retherapy. Conclusion: With careful exclusion of organic diseases other than HP gastritis, dyspeptic patients can be selected who clearly benefit from eradication therapy. G0471
SUCCESSFUL VS "LESS SUCCESSFUL" FUNDOPLICATIONS: TOO LOOSE? TOO TIGHT? TOO LOW? GA Er~un, S Lin, RJ Joehl, PJ Kahrilas. Depts of Med & Surgery, Northwestern Univ, Chicago, IL. Antireflux surgery and antisecretory therapy are established, effective remedies for erosive esophagitis. Accordingly, a population of medically "refractory patients" has emerged, without esophagitis, but with symptoms believed related to reflux. We examined the symptom profile as well as functional characteristics of the gastroesophageal junction (GEJ) in such patients who opted for antireflux surgery and who were either satisfied or not satisfied with their surgery. Methods: Synchronized videofluoroscopic and esophageal manometric studies were performed on 7 asymptomatic, herniafree controls and 14 post-laparoscopic Nissen fundoplication patients (7 successes (Fundo), 7 with post-surgery symptoms (SxFundo)) during transit of a 10 ml liquid bolus and a 1.3 cm marshmallow after endoscopic evaluation and placement of a metal clip at the squamocolumnar junction. None were done for refractory erosive esophagitis. Parameters examined were completeness of LES relaxation (Nadir LESR), intrabolus pressure at the GEJ during liquid emptying (Amp IBP), intrabolus pressure in the esophagus during liquid transit (Esoph IBP), maximal luminal width within the hiatus during transit (Hiatal width), length of the wrap (Wrap length), and axial excursion of the squamocolumnar junction during peristalsis (Axial mvmnt). Results: All of the Sx Fundo group reported a 1-8 week symptom-free period; subsequent symptoms reported were heartburn (4), regurgitation (4), chest pain (5), dysphagia (2), and miscellaneous (2). 5/7 Sx fundo patients resumed taking antisecretory medications (2 at a lesser dose than preop, the others equal). Marshmallow transit time across the GEJ was markedly prolonged in both Fundo (3 required multiple swallows) and SxFundo patients (4 required multiple swallows). Several functional abnormalities could be demonstrated in the Fundo group and these abnormalities tended to be accentuated in the SxFundo group (Table). Nadir Amp Esoph Hiatal Wrap Axial Group LESR§ IBP§ IBP§ widths length~: mvmnt~ Control 4 +-3 24 -+3 8 -+ 1 1.8 -+ .4 .... 2.9 -+.5 Fundo 6_+3 38_+5* 11_+2 0.8_+.1" 3.4_+.2 1.3_+.2" SxFundo ll_+l*t 35_+4* 17_+2"t 0.6_+.l*t 3.7_+.3 0.9_+.l*t § mean -+SD mmHg, :~mean +-SD cm, *p < .05 vs control, t P< .05 vs Fundo
• G0468 A NOVEL
REAL-TIME QUANTITATIVE PCR ASSAY FOR DETECTION OF HELICOBACTER PYLORI IN THE GASTRIC MUCOSA. M. Endoh, T. Shimada, N. Watanabe, Y. Ohtsuka, K. Sakata, T. Sasai, H. Hiraishi, A. Terano. Second Department of Internal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0207, Japan. BACKGROUND: Although the assessment of the density of Helicobacter pylori (H. pylori) colonization may be important, precise quantitative evaluation of H. pylori in the gastric mucosa has been difficult. We have developed a novel real-time quantitative PCR technique based on 5' -3' exonuclease assay for detection of H. pylori. Using this system, we evaluated number of H. pylori organisms present in gastric biopsy specimens. METHODS: A primer pair for PCR amplification was made according to the reported sequence of ureA gene of H. pylori, and an oligonucleotide probe labeled with two fluorescent dyes (a reporter dye at the 5' end and a quencher dye at the 3' end) was also designed. While the probe is intact, the quencher dye greatly reduces the fluorescent signal by the reporter dye (Fluorescence resonance energy transfer phenomenon). During the PCR extension phase, the annealed probe is cleaved by 5' -3' exonuclease activity of Taq DNA polymerase, increasing the reporter dye signal. The reporter dye signal during a PCR assay was monitored with a ABI PRISM 7700 Sequence Detector (Perkin-Elmer). Purified PCR products of ureA gene were used as standard samples for making standard curves plotting log starting copy number and threshold cycle (Ct: PCR cycle number at which the reporter fluorescence signal reached above baseline). Biopsy specimens were collected from antral mucosa of consecutive 32 patients underwent gastroscopy (GU 9, DU 9, GC 1, NUD 13). Three biopsy specimens were obtained from each patient to avoid the effect of patchy distribution of H. pylori. DNA was extracted by conventional phenol-chloroform method. Since the size of each biopsy specimen was variable, quantitative PCR assay described above was performed using 0.5 ~tg of DNA extracted from each sample. !~_!LL_.T~: In each amplification reaction, a liner standard curve could be generated in a wide range of starting copy numbers (6xl07 to 6xl0 copies of the target sequences, R2 > 0.99). Nine of 32 samples (DU 2, NUD 7) showed no increase in the fluorescence signal during PCR process, indicating the absence of H. pylori, which was also confirmed by histological examinations. In H. pylori positive patients, the detected ureA copy numbers (i.e. number of H. pylori organisms) were variable among patients (range 1.4x102 - 1.2x105 copies/0.5Mg DNA, mean 2.6x104 copies/0.51ag DNA). The average ureA copy numbers of samples from DU and GU patients were not significantly different from that of NUD patients [GU (2.1 _+ l.l)xl04, DU (1.8 _+0.6)xl04, NUD (2.6 + 1.1)xl04 copies/0.51ag DNA, respectively]. The sample of one GC patient showed high density of H. pylori colonization (1.2x105 copies/0.5Mg DNA). CONCLUSIONS: Accurate quantitation of H. pylori in biopsy specimens was possible with this PCR system, and it will be useful for 1-1.pylori research. G0469
GLUCOSE INFUSIONS INTO STOMACH OR JEJUNUM DECREASE GASTRIC INJURY FROM RESTRAINT STRESS. K. Ephgrave, A. Ong, K. Broadhurst, J. Cullen. VAMC and University of Iowa Hospital College of Medicine, Iowa City, Iowa. Intragastric 25% glucose (D25) at 2ml/hr during restraint stress prevents mucosal injury, increases intragastric pH, and suppresses stress-induced hypercontractility in rats. Intrajejunal glucose was equally protective at this rate, but with glucose reflux into the stomach. The purpose of the present study was to test the hypothesis that enteral glucose can initiate gastric protection without contacting gastric afferents. Methods: Male 200-300 gm Sprague-Dawley rats were prepared with gastric, jejunal, and jugular catheters, and a strain gauge transducer on the anterior surface of the gastric antrum. After 5 days recovery, they underwent a restraint protocol at room temperature for 2 hours followed by cold (4 °) for an additional 2 hours, while receiving .5 ml/hr saline IG (n=6), D25 IG (n=6), D25 IJ (n=6), or saline IJ (n=3). Gastric lesion length, volume, pH, glucose, and mucin were measured following stress. Gastric motility was measured continuously and blood glucose was measured terminally. Findings: Lesion length (mm) decreased in both of the groups receiving D25, (IG saline 10.1 -+ 1.8, IG D25 4.0 _+ 1.4", IJ D25 2.5 _+ 1.0", IJ saline 5.7 _+ 1.4, *p < .01 vs. saline). No effect was seen on gastric pH (IG saline 2.2_+.2, IG D25 2.1 _+.3, IJ D25 2.1 _+.2, IJ saline 1.9 _+.1), nor mucus (29.7 _+4 vs. 25.1 _+9 U, IJ D25 vs. IJ saline). Stressinduced gastric hypercontractility regressed for < 10 minutes following IG D25, but then recurred. No hyperglycemia occurred, and no jejunal glucose refiuxed intragastrically, as gastric levels were 611 + 28 mg/dl for IG D25 versus 67 + 11 forlJ D25, p < .01. Conclusions: Small volumes of jejunal or gastric glucose ameliorate restraintinduced gastric injury, though jejunal infusion does not affect gastric glucose levels. The infusions did not affect gastric contractions, acidity, or mucin production, so the protective mechanism is unknown. Study supported by VA Merit Review Funds
G0470
EFFECT OF ERADICATION ON DYSPEPTIC SYMPTOMS OF HELICOBACTER PYLORIoPOSITIVE PATIENTS. H.J. Epple 1, C. Bojarski 1,2, F.W. Kirstein 1, M. Fromm2, E.O. Riecken l, J.D. SchulzkeL Depts. of ]Gastroenterology & Infectiology and 2Clinical Physiology, UKBF, Freie Universit~it Berlin, D- 12200 Berlin, Germany The subgroup of patients with dyspepsia for whom eradication therapy can be recommended has not been defined so far. In this study, we assessed the effect of Helicobacter pylori (HP) eradication on dyspeptic symptoms of a highly selected group of HP positive patients. re*0:
7o.
m,
First treatment
~zo~~a°~ E successf(n=l ul O)
unlriel thempy (n=7)
pemieten4H P-infecti~
10
before'therapy
altethrerapy
therapy
therapy
retherapy
Inclusion criteria were dyspepsia for at least 6 months, HP infection (diagnosed by laC-urea breath test (UBT) and histology), no active or previous peptic ulcer. After gastroscopy the patients underwent abdominal sonography, H2-exhalation test with lactose, and 24 h oesophageal pHmonitoring in order to exclude organic diseases causing dyspepsia. The remaining patients received eradication therapy. Eradication was confirmed by 13C-UBT 6 weeks after completion of the therapy. The severity of dyspeptic symptoms was monitored with a validated score in a blinded fashion before and 6 months after therapy or retherapy, respectively. From an initial number of 128 patients 87 patients were excluded after endoscopy and the other investigations. 24 patients refused additional investigations after the endoscopy. 17 patients without other organic disease except for HP gastritis were treated with eradication therapy. In 10 patients the first treatment was successful, while 7 patients were HP-negative only after retberapy. The symptoms of successfully treated patients improved considerably (see Figs.), whereas symptoms of patients with persistent infection remained unchanged (AS15 vs A0, p < 0.05) and only improved after successful retherapy. Conclusion: With careful exclusion of organic diseases other than HP gastritis, dyspeptic patients can be selected who clearly benefit from eradication therapy. G0471
SUCCESSFUL VS "LESS SUCCESSFUL" FUNDOPLICATIONS: TOO LOOSE? TOO TIGHT? TOO LOW? GA Er~un, S Lin, RJ Joehl, PJ Kahrilas. Depts of Med & Surgery, Northwestern Univ, Chicago, IL. Antireflux surgery and antisecretory therapy are established, effective remedies for erosive esophagitis. Accordingly, a population of medically "refractory patients" has emerged, without esophagitis, but with symptoms believed related to reflux. We examined the symptom profile as well as functional characteristics of the gastroesophageal junction (GEJ) in such patients who opted for antireflux surgery and who were either satisfied or not satisfied with their surgery. Methods: Synchronized videofluoroscopic and esophageal manometric studies were performed on 7 asymptomatic, herniafree controls and 14 post-laparoscopic Nissen fundoplication patients (7 successes (Fundo), 7 with post-surgery symptoms (SxFundo)) during transit of a 10 ml liquid bolus and a 1.3 cm marshmallow after endoscopic evaluation and placement of a metal clip at the squamocolumnar junction. None were done for refractory erosive esophagitis. Parameters examined were completeness of LES relaxation (Nadir LESR), intrabolus pressure at the GEJ during liquid emptying (Amp IBP), intrabolus pressure in the esophagus during liquid transit (Esoph IBP), maximal luminal width within the hiatus during transit (Hiatal width), length of the wrap (Wrap length), and axial excursion of the squamocolumnar junction during peristalsis (Axial mvmnt). Results: All of the Sx Fundo group reported a 1-8 week symptom-free period; subsequent symptoms reported were heartburn (4), regurgitation (4), chest pain (5), dysphagia (2), and miscellaneous (2). 5/7 Sx fundo patients resumed taking antisecretory medications (2 at a lesser dose than preop, the others equal). Marshmallow transit time across the GEJ was markedly prolonged in both Fundo (3 required multiple swallows) and SxFundo patients (4 required multiple swallows). Several functional abnormalities could be demonstrated in the Fundo group and these abnormalities tended to be accentuated in the SxFundo group (Table). Nadir Amp Esoph Hiatal Wrap Axial Group LESR§ IBP§ IBP§ widths length~: mvmnt~ Control 4 +-3 24 -+3 8 -+ 1 1.8 -+ .4 .... 2.9 -+.5 Fundo 6_+3 38_+5* 11_+2 0.8_+.1" 3.4_+.2 1.3_+.2" SxFundo ll_+l*t 35_+4* 17_+2"t 0.6_+.l*t 3.7_+.3 0.9_+.l*t § mean -+SD mmHg, :~mean +-SD cm, *p < .05 vs control, t P< .05 vs Fundo