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Role of Helicobacter pylori in the pathogenesis of hyperammonemia in the liver cirrhotic rats
A148
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 1 0 8 , No. 4
• DETECTION OF H. PYLORI DNA IN SALIVA AS A MEANS OF MONITOR/NG ERADICATION THERAPY C. Li 1, T. Ha1, DA. Ferguson, jr2., RG. Zhao, DS. Chi ~, and E. Thomas 1'3 Departments of Internal Medicine I and Microbiology 2, James H. Quillen College of Medicine, East Tennessee State University and VA Medical Center 3, Johnson City, TN 37684 The oral cavity is attracting increasing attention as a reservoir of H. .pylori infection and transmission. Detection of H. pylori DNA in the oral cavity would provide a simple, rapid, precise, and non-invasive method for diagnosing and monitoring the efficacy of eradication therapy. In this study, a PCR assay was employed to monitor the efficacy of eradication therapy of H. pylori infection by serially detecting and tracking H. pylori DNA in saliva. Twelve subjects were involved in this study. They all had experienced many years of dyspepsia. H. pylori infection was confirmed by endoscopy (CLO test & stain) in 8 and by ELISA (IgG antibody) in 4. Saliva specimens were collected directly into sterile containers containing digestion buffer. H. pylori DNA was identified by PCR assay in the saliva from seven of the 12 patients and confirmed by Southern blot hybridization and restriction enzyme analysis. Six of the 7 patients, whose saliva showed H. pylori DNA, received eradication therapy and one did not. H. pylori DNA continued to be detected in the saliva two weeks post-treatment in two of the 6 patients. Between three weeks and 3 months post-treatment, the other four patients experienced relief of their chronic gastroduodenal symptoms coincident with the disappearance of H. pylori DNA from their saliva. Conversely, H. pylori-specific DNA was consistently detected in the saliva of the one untreated subject for over 3 months. These observations indicate that detection of H. pylori DNA in saliva by PCR may be useful for monitoring and managing treatment.
• GASTRIN INCREASES CELL PROLIFERATION IN GASTRODUODENAL EPITHELIUM DURING HEALING OF GASTRIC ULCER IN RAT. H Li, I-IFHelander, Astra H~issle AB~ M61ndal. Sweden. Aim: To determine if gastrin influences the proliferation of the gastroduodenal epithelium during healing of experimental gastric ulcers. Methods: In 3 groups of rats (9-11 per group), an ulcer was induced in the oxyntic mucosa by the acetic acid method. Immediately after the operation, one group was given vehicle (0.5% Methocel®, once daily¢ p.o), another group omeprazole (400 ~mol/kg, once daily, p.o) and the last group gastrin-17 (60 nmol/kg/day, sc. by osmotic minipumps). Half of the rats (4-6 per group) were killed after 3 days and the remaining ones after 6 days. Unoperated rats given vehicle served as controls. One hr before sacrifice, each rat was given an injection of 3H-thymidine. Fixation by vascular peffusion of 4% formaldehyde Was preceeded by blood sampling for gastdn analysis. Tissue samples from the corpus (including the ulcer), the antrum and the duodenum were embedded in plastic. Twopm sections were cut and processed for autoradiography. Results: Immediately before sacrifice, the plasma gastrin levels were 5 times higher in the omeprazole treated rats (24 hrs after last dose) than in vehicle treated ulcer rats; in the rats given gastrin-17, the mean increase was 14 times. The mean labelling index (LI) of the oxyntic epithelium in the intact controls was 1.9+0.4%. In the ulcer rats, mean LI in the ulcer margin epithelium of the vehicle treated rats was 8.7±0.5%; the corresponding figure in the omeprazole treated groups was 10.9-+0.6% and in the gastrin-17 treated groups 11.6+0.6%. In the duodenal crypts of the vehicle treated ulcer rats, LI was 42.4+2.5% i vs 27.7+L7% in the vehicle treated intact controls. In the ulcer rats, no significant changes were registered in the number of epithelial cells per rrnn2 or mucosal thickness of the corpus (distant from the ulcer), antrum or duodenum, when compared with the unoperated controls. Comment: The augmented LI in the ulcer margin following omeprazole and gastrin-17 might be due to the hypergastrinemia and this might increase the rate of ulcer healing. It remains to determine the cause for the increased LI in the duodenal crypts of the ulcer rats.
• INHIBITION BY PACAP OF GASTRIC ACID SECRETION iS MEDIATED BY SECRETIN, SOMATOSTATIN AND PGE2 IN ISOLATED PERFUSED RAT STOMACH. P.LI, T-M CHANG, "D Coy, W.Y. Chey. University of Rochester Medical Center, Rochester, NY and "Tulane Medical Center, NewOdeans, LA.
O ROLE OF Helicobacter pylori IN THE PATHOGENESIS OF HYPERAMMONEMIA IN THE LIVER CIRRHOTIC RATS. Y.M. Li.* S. Ito, Y. Kol'di, Y. Ishii. The Second Department of Internal Medicine, Fukul Medical School, Fukui, Japan. *Zhejiang Medical School, China
Pituitary adenylate cyclase activating peptide (PACAP) is found in neural elements throughout the GI tract, but the effect of PACAP on gastric acid secretion and its mechanism remains udclear. To investigate the inhibitory effect of PACAP and its mechanism on gastric acid secretion, totally isolated rat stomachs were vasculady peffused with Krebs-Ringer buffer containing 10% fresh rat erythrocytes, 4% bovine albumin and 50 p.M isobutyl methylxanthir'e (IMX) at 1.4 mVmin, The perfusate was maintained at 3"f C and gassed with 95% O 2 and 5% CO2. The gastric lumen was perfused with 0.15 N NaCI at 1.0 ml/min. Both basal and pentagastdn (50 ng/h)-stimulated gastric acid secretion were dose-dependently inhibited by PACAP given intra-artedally (ia) at 5,10, and 20 p.g/h, respectively. To determine possible paracdne mediation in PACAP-inhibited acid secretion, secretin (SEC), somatostatin (SS) and prostaglandin ~ (PGE2) were measured in portal venous effluents by RIA from 3 groups of rats (5-11). Basal Sec. SS and PGEz concentrations were 0.36:L-0.1 pM, 14,1+9.4 pM and 286.2.t:88.7 pM, respectively. PACAP (10 p.g/h) significantly increased SEC, SS and PGE2 levels to 3.8i-0.7 pM (p<0.05), 48.7:L--6.7pM (p<0.05), and 1504.4+411.1 pM (p<0.05), respectively. The inhibitory effect of PACAP (10 p.g/h) on the pentagastdn-stimulated acid secretion was reversed by a rabbit antisomatostatin serum (69.8%), indomethecin (46.1%) or a rabbit antisecretin serum (33%) respectively; whereas normal rabbit serum did not influence the inhibition by PACAP (10 p.g/h), Rabbit antisecretin serum also partially reduced PACAP (10 p.g/h)-induced SS and PGEz increase in portal venous effluent from 48.7i.6.7 pM to 30.2:1:5.1 pM and 1504,4+411.1 pM to 692.8:1:207,2 pM, respectively, It is suggested that PACAP-induced inhibition of both basal and pentagastdn-stimulated gastdc acid secretion are partially mediated by local increases in Sec, SS and PGEz in isolated perfused rat stomach. The increase in SS and PGF-= may, in part, be due to secretin.
Helicobacter pylori is involved not only in gastroduodenal mucosal diseases, but also in production of gastric ammonia that is generated from the hydrolysis of urea, a reaction catalyzed by the ureas¢ enzyme of H.pylorL In the present study, vce investigated the influence of gastric ammonia produced by H.pylori on the blood ammonia levels in the presence of decompensative and compensative liver cirrhosis, in order to clarify the role of H.pylori in the pathogenesis of hyperammonemia. Using an animal model of liver cirrhosis induced by injection of carbon tetrachloride, we instilled directly lml live H.pylori suspension and lm1400 mmol/L urea into the stomach of cirrhotic or normal rats with disposable insulin syringes. As control groups, 2ml of sterile saline was similarly instilled in cirrhotic or normal rats. Venous blood from the right femoral vein and portal blood from the proximal portion of the superior mesenteric vein or portal vein itself were obtained at 0, 15, 30, 60, 120, 180 min. Gastric juice was obtained at 0 and I20 rain from each group. Blood ammonia and gastric ammonia were quantitated using an Amicheckmeter. Instillation of saline into the Stomachof cirrhotic rats did not result in any significant change in the venous and portal ammonia levels, while instillation of live H. pylori suspension resulted in a marked elevation in the blood ammonia levels after 30 min. After 120 min, the venous and portal ammonia levels reached 109 and 615 p.mol/L, respectively. In addition, the degree of elevation was greater in the decompensative cirrhotic rats compared with the compensative cirrhotic rats, with the venous blood ammonia levels of median 134 (range 112~138)p.mol/L and 103.5(range 64-109)lxmol/L (p=0.034). The portal blood ammonia levels in the decompensative cirrhotic rats were almost equal to that of the compensative ones (p--0.724). The gastric ammonia levels in the cirrhotic rats at 120 min were about 1.8x105 ~tmol/L, which was not significantly differrent (p=0.668), compared with the normal group. This result suggests that the ammonia produced by H.pylori in the stomach contributed significantly to blood ammonia level, particularly in the presence of liver cirrhosis, if this organism is present in large number. In conclusion, the ammonia produced by Helicobacter pylori in the stomach may play an important role in the pathogenesis of hyperammonemia in the presence of liver cirrhosis, particularly in the decompensativ¢ state. We believe that further investigation is necessary for the relation of infection with Helicobacterpylori in the gastric mucosa to hepatic encephalopathy in human beings.
AGA ABSTRACTS
GASTROENTEROLOGY, Vol. 1 0 8 , No. 4
• DETECTION OF H. PYLORI DNA IN SALIVA AS A MEANS OF MONITOR/NG ERADICATION THERAPY C. Li 1, T. Ha1, DA. Ferguson, jr2., RG. Zhao, DS. Chi ~, and E. Thomas 1'3 Departments of Internal Medicine I and Microbiology 2, James H. Quillen College of Medicine, East Tennessee State University and VA Medical Center 3, Johnson City, TN 37684 The oral cavity is attracting increasing attention as a reservoir of H. .pylori infection and transmission. Detection of H. pylori DNA in the oral cavity would provide a simple, rapid, precise, and non-invasive method for diagnosing and monitoring the efficacy of eradication therapy. In this study, a PCR assay was employed to monitor the efficacy of eradication therapy of H. pylori infection by serially detecting and tracking H. pylori DNA in saliva. Twelve subjects were involved in this study. They all had experienced many years of dyspepsia. H. pylori infection was confirmed by endoscopy (CLO test & stain) in 8 and by ELISA (IgG antibody) in 4. Saliva specimens were collected directly into sterile containers containing digestion buffer. H. pylori DNA was identified by PCR assay in the saliva from seven of the 12 patients and confirmed by Southern blot hybridization and restriction enzyme analysis. Six of the 7 patients, whose saliva showed H. pylori DNA, received eradication therapy and one did not. H. pylori DNA continued to be detected in the saliva two weeks post-treatment in two of the 6 patients. Between three weeks and 3 months post-treatment, the other four patients experienced relief of their chronic gastroduodenal symptoms coincident with the disappearance of H. pylori DNA from their saliva. Conversely, H. pylori-specific DNA was consistently detected in the saliva of the one untreated subject for over 3 months. These observations indicate that detection of H. pylori DNA in saliva by PCR may be useful for monitoring and managing treatment.
• GASTRIN INCREASES CELL PROLIFERATION IN GASTRODUODENAL EPITHELIUM DURING HEALING OF GASTRIC ULCER IN RAT. H Li, I-IFHelander, Astra H~issle AB~ M61ndal. Sweden. Aim: To determine if gastrin influences the proliferation of the gastroduodenal epithelium during healing of experimental gastric ulcers. Methods: In 3 groups of rats (9-11 per group), an ulcer was induced in the oxyntic mucosa by the acetic acid method. Immediately after the operation, one group was given vehicle (0.5% Methocel®, once daily¢ p.o), another group omeprazole (400 ~mol/kg, once daily, p.o) and the last group gastrin-17 (60 nmol/kg/day, sc. by osmotic minipumps). Half of the rats (4-6 per group) were killed after 3 days and the remaining ones after 6 days. Unoperated rats given vehicle served as controls. One hr before sacrifice, each rat was given an injection of 3H-thymidine. Fixation by vascular peffusion of 4% formaldehyde Was preceeded by blood sampling for gastdn analysis. Tissue samples from the corpus (including the ulcer), the antrum and the duodenum were embedded in plastic. Twopm sections were cut and processed for autoradiography. Results: Immediately before sacrifice, the plasma gastrin levels were 5 times higher in the omeprazole treated rats (24 hrs after last dose) than in vehicle treated ulcer rats; in the rats given gastrin-17, the mean increase was 14 times. The mean labelling index (LI) of the oxyntic epithelium in the intact controls was 1.9+0.4%. In the ulcer rats, mean LI in the ulcer margin epithelium of the vehicle treated rats was 8.7±0.5%; the corresponding figure in the omeprazole treated groups was 10.9-+0.6% and in the gastrin-17 treated groups 11.6+0.6%. In the duodenal crypts of the vehicle treated ulcer rats, LI was 42.4+2.5% i vs 27.7+L7% in the vehicle treated intact controls. In the ulcer rats, no significant changes were registered in the number of epithelial cells per rrnn2 or mucosal thickness of the corpus (distant from the ulcer), antrum or duodenum, when compared with the unoperated controls. Comment: The augmented LI in the ulcer margin following omeprazole and gastrin-17 might be due to the hypergastrinemia and this might increase the rate of ulcer healing. It remains to determine the cause for the increased LI in the duodenal crypts of the ulcer rats.
• INHIBITION BY PACAP OF GASTRIC ACID SECRETION iS MEDIATED BY SECRETIN, SOMATOSTATIN AND PGE2 IN ISOLATED PERFUSED RAT STOMACH. P.LI, T-M CHANG, "D Coy, W.Y. Chey. University of Rochester Medical Center, Rochester, NY and "Tulane Medical Center, NewOdeans, LA.
O ROLE OF Helicobacter pylori IN THE PATHOGENESIS OF HYPERAMMONEMIA IN THE LIVER CIRRHOTIC RATS. Y.M. Li.* S. Ito, Y. Kol'di, Y. Ishii. The Second Department of Internal Medicine, Fukul Medical School, Fukui, Japan. *Zhejiang Medical School, China
Pituitary adenylate cyclase activating peptide (PACAP) is found in neural elements throughout the GI tract, but the effect of PACAP on gastric acid secretion and its mechanism remains udclear. To investigate the inhibitory effect of PACAP and its mechanism on gastric acid secretion, totally isolated rat stomachs were vasculady peffused with Krebs-Ringer buffer containing 10% fresh rat erythrocytes, 4% bovine albumin and 50 p.M isobutyl methylxanthir'e (IMX) at 1.4 mVmin, The perfusate was maintained at 3"f C and gassed with 95% O 2 and 5% CO2. The gastric lumen was perfused with 0.15 N NaCI at 1.0 ml/min. Both basal and pentagastdn (50 ng/h)-stimulated gastric acid secretion were dose-dependently inhibited by PACAP given intra-artedally (ia) at 5,10, and 20 p.g/h, respectively. To determine possible paracdne mediation in PACAP-inhibited acid secretion, secretin (SEC), somatostatin (SS) and prostaglandin ~ (PGE2) were measured in portal venous effluents by RIA from 3 groups of rats (5-11). Basal Sec. SS and PGEz concentrations were 0.36:L-0.1 pM, 14,1+9.4 pM and 286.2.t:88.7 pM, respectively. PACAP (10 p.g/h) significantly increased SEC, SS and PGE2 levels to 3.8i-0.7 pM (p<0.05), 48.7:L--6.7pM (p<0.05), and 1504.4+411.1 pM (p<0.05), respectively. The inhibitory effect of PACAP (10 p.g/h) on the pentagastdn-stimulated acid secretion was reversed by a rabbit antisomatostatin serum (69.8%), indomethecin (46.1%) or a rabbit antisecretin serum (33%) respectively; whereas normal rabbit serum did not influence the inhibition by PACAP (10 p.g/h), Rabbit antisecretin serum also partially reduced PACAP (10 p.g/h)-induced SS and PGEz increase in portal venous effluent from 48.7i.6.7 pM to 30.2:1:5.1 pM and 1504,4+411.1 pM to 692.8:1:207,2 pM, respectively, It is suggested that PACAP-induced inhibition of both basal and pentagastdn-stimulated gastdc acid secretion are partially mediated by local increases in Sec, SS and PGEz in isolated perfused rat stomach. The increase in SS and PGF-= may, in part, be due to secretin.
Helicobacter pylori is involved not only in gastroduodenal mucosal diseases, but also in production of gastric ammonia that is generated from the hydrolysis of urea, a reaction catalyzed by the ureas¢ enzyme of H.pylorL In the present study, vce investigated the influence of gastric ammonia produced by H.pylori on the blood ammonia levels in the presence of decompensative and compensative liver cirrhosis, in order to clarify the role of H.pylori in the pathogenesis of hyperammonemia. Using an animal model of liver cirrhosis induced by injection of carbon tetrachloride, we instilled directly lml live H.pylori suspension and lm1400 mmol/L urea into the stomach of cirrhotic or normal rats with disposable insulin syringes. As control groups, 2ml of sterile saline was similarly instilled in cirrhotic or normal rats. Venous blood from the right femoral vein and portal blood from the proximal portion of the superior mesenteric vein or portal vein itself were obtained at 0, 15, 30, 60, 120, 180 min. Gastric juice was obtained at 0 and I20 rain from each group. Blood ammonia and gastric ammonia were quantitated using an Amicheckmeter. Instillation of saline into the Stomachof cirrhotic rats did not result in any significant change in the venous and portal ammonia levels, while instillation of live H. pylori suspension resulted in a marked elevation in the blood ammonia levels after 30 min. After 120 min, the venous and portal ammonia levels reached 109 and 615 p.mol/L, respectively. In addition, the degree of elevation was greater in the decompensative cirrhotic rats compared with the compensative cirrhotic rats, with the venous blood ammonia levels of median 134 (range 112~138)p.mol/L and 103.5(range 64-109)lxmol/L (p=0.034). The portal blood ammonia levels in the decompensative cirrhotic rats were almost equal to that of the compensative ones (p--0.724). The gastric ammonia levels in the cirrhotic rats at 120 min were about 1.8x105 ~tmol/L, which was not significantly differrent (p=0.668), compared with the normal group. This result suggests that the ammonia produced by H.pylori in the stomach contributed significantly to blood ammonia level, particularly in the presence of liver cirrhosis, if this organism is present in large number. In conclusion, the ammonia produced by Helicobacter pylori in the stomach may play an important role in the pathogenesis of hyperammonemia in the presence of liver cirrhosis, particularly in the decompensativ¢ state. We believe that further investigation is necessary for the relation of infection with Helicobacterpylori in the gastric mucosa to hepatic encephalopathy in human beings.