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New method for characterization of mucin in inflammatly bowel disease using whole gut lavage fluid
April 1995
Immunology, Microbiology, and Inflammatory Disorders A909
O A DOUBLE-BLIND COMPARISON OF ORAL VERSUS RECTAL MESALAMINE VERSUS COMBINATION THERAPY IN THE TREATMENT OF DISTAL ULCERATIVE COLITIS. The Rowasa® Study Group: M. Safdi, M. DeMicco, C. Sninsky, P. Banks, L. Wruble, J. Deren, G. Koval, T. Nichols, S. Targan, D. Taylor, C. Fleishman, T. Nester. (Sites in OH, CA, FL, MA, TN, PA, OR). A 6-week, multieanter, randomized, double-blind trial was performed in 60 patients with active, histologically confirmed, mild to moderate distal ( ~ 50 era) ulcerative colitis (UC) to evaluate the efficacy and safety of oral (O) mesalamine tablets 800rag tid (n=22) vs. a 4g rectal (R) mesalamine enema qhs (n= 18) vs. the combination (C) of oral and rectal therapy (n=20). After baseline, patients were evaluated at weeks 1, 2, 3 and 6 with endoscopic evaluations at weeks 3 and 6. Efficacy evaluations included a disease activity index (DAD and a 7-point physician and patient clinical global impression (CGI) rating. The DAI represents a composite score of four subscales: evacuation frequency, rectal bleeding, mucosal appearance and physician's overall assessment of disease severity. Patient demographics, including UC history, did not differ significantly among treatment groups. Improvement in total DAI score was greater for C (-5.3 points) followed by R (-3.9) and O (-3.4) but this trend did not reach statistical significance. The subscale measuring rectal bleeding status was significantly different among treatments at weeks I, 2 and 6 (see table): Treatment % PatientsWithout Physician mean CGI scores revealed greater Period RectalBleeding improvement (p < 0.05) with C compared to O at all visits and R compared to O at weeks 2, 3 o R c and 6. At final assessment the percentage of Week 1 9 33* 40* patients rated by their physician as much or very much improved was 45, 78 and 89 for O, Week 2 lS 35 70* R and C, respectively. Greater improvement Week 3 43 5# 75 (P < 0.05) in patient CGI scores was observed for R compared to 0 and C compared to 0 at Week 6 36 56 95** weeks 2, 3 and 6. The percentage of patients * p
NEW METHOD FOR CHARACTERIZATION OF MUCIN IN INFLAMMATLY BOWEL DISEASE USING WHOLE GUT LAVAGE FLUID.
O SERUM BILE ACIDS, PLASMA CHOLECYSTOKININ (CCK), AND GALLBLADDER MOTILITY AFTER ILEAL RESECTION (IR). J.MJ.I. Salem~n~, F.M. Nagengast, A. van Schaik, A. Tangerman, W.P.M. Hopman, J.B.M.J. Jansen. Dept. of Gastroenterology, University Hospital Nijmegen, The Netherlands. IR is associated with bile acid malabsorption and an increased risk for cholelithiasis. A decreased duodenal bile acid output and increased biliary ursodeoxycholic acid (UDCA) fraction have been found after IR. The aim of this study was to examine whether IR leads to alterations in bile acid absorption, deconjugation, 7~-dehydroxylation, and formation of UDCA. Since intraduodenal bile acids may influence the release of CCK we further studied the effect of IR on plasma CCK and gallbladder volume (GBV). Methods: 8 patients with Crohn's disease and a history of IR (M/F=5/3, mean age 39, operation 1-14 yrs before this study, length of IR: 30-70 cm) and 12 healthy controls (M/F=8/4, mean age 43) were studied. None of the subjects were using cholestyramine. Fasting and postprandial blood samples were obtained at 15 rain intervals (3 hours) for conjugated (c) and unconjugated (unc) cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), and UDCA (gas-liquid chromatography) and CCK (RIA). GBV was measured using ultrasonography. Results: Controls IR Integrated serum bile acids (p.M. 180min) CA (c) 1854-26 83+32 p < 0.005 CDCA (c) 4544-67 2764-43 p <0.05 DCA (c) 194_+25 36_+5 p < 0.0001 UDCA (c) not detected not detected CA (unc) 44+6 974-23 p<0.05 CDCA (uric) 60+7 233_+51 p<0.01 DCA (unc) 65 _+9 56_+7 NS UDCA (unc) not detected 144+54 p<0.01 Fasting GBV (mL) 28 + 3 27 _+7 NS Percentage GB emptying (%) 78+ 3 83 +5 NS Fasting plasma CCK (pM) 3.4+0.2 3.54-0.4 NS Integrated CCK response (pM.180min) 898+73 961+ 130 NS Conclusions: postprandial conjugated bile acid levels are decreased while unconjuguted bile acid levels are increased after IR. Formation of DCA is reduced .after IR, probably as a result of rapid colonic transit with shortened exposure to bacterial 7~-dehydroxylase. The production of UDCA is increased after IR, which may be due to CDCA malabsorption. Bile acid malabsorption after IR does not lead to alterations in CCK release and gallbladder motility.
INC~ED SERINE DEPENDENT PROTEINASES IN ULCERATIVE COLITIS: MUCOLYSIS AND INHIBITION BY DIOSMECTITE H.J. Samson, J.P. Pearson, E.D. Srivastava, *M-T Droy-Lefalx and A. Allen, Department of Physiological Sciences, University of Newcastle upon Tyne, UK, and *Department of Pharmacology, Ipsen Institute, Paris, France.
H. Saitoh, Y. Uno, H. Nakajima, A. Munakata, M. Endo and Y. Yoshida. First Depts. of Internal Medichine & Biochemistry, Hirosaki University School of Medicine, Hirosaki, Japan Whole gut lavage fluid (WGLF) obtained using polyethyleneglycol-based orally administered balanced electrolyte solution prior to colonoscopy was examined in order to investigate the biochemical features of intestinal mucin in 20 normal subjects and 12 patients with inflammatory bowel disease (IBD) (5 with Crohn's disease (CD) and 7 with ulcerative colitis (UC)). Methods: Each WGLF sample was chromatographed on a gel-filtration column of Sepharose CL-4B, and then Sepharose CL-2B. Quantities of the mucin recovered in the void volume were then estimated. Ultracentrifugation was carried out to purify the mucin, followed by ion-exchange chromatography to separate neutral and acidic mucins. Results: In UC, the amount of mucin was decreased, whereas in CD, the amount of mucin was increased in comparison with normal subjects (p<0.05). Furthermore, the mucin acidity was estimated in terms of the acidic mucin/neutral mucin ratio (A/N). UC patients had a higher A/N than normal subjects (p<0.05).
subjects
l
[ normal
~
amount of mucin (mean±SD) mg/500 ml 14.99-18.13
(16.78)
10.s4-13.26 (11.971.
17.68-25.45 (20.89)*
A/N(mean±SD)
1.23-1.39(1.35)
2.87-3.69(3.2s)
i.Ii-1.42(i.14)
*p<0.05 vs. control Conclusion: The WGLF method is useful for investigation of mucin, and may reveal pathological changes in the mucosa in IBD.
Patients with ulcerative colitis (UC) have significantly raised total faecal proteinase activity compared to healthy controls. Using ion exchange HPLC faecal proteinases were ffactionated into two peaks of proteinase activity: pools I and 2. Pool 1 was eluted before application of a salt gradient and was coincident with 98% of pancreatic proteinase activity; pool 2 einted during the salt gradient. Mean proteinase activity (expressed in units of mmoles NI~ terminals min'lg"xdry weight of faeces, U) for pool 2 was significantly, six-fold higher for UC patients (6.8 5: 2.2U, n=9) than that for controls (1.2 5: 0.5U n = 10) p < 0.05. Mean proteinase activity for pool 1 from UC patients (4.6 5: 2.1U n = 9 ) was also six-fold greater than that for controls (0.7 5: 0.2U n = 10) although not significant. Specific inhibition showed that serine dependent proteinases were the major component ( > 76%) in pools 1 and 2 from both controls and UC patients. Diosmectito, a non-systemic, antidiarrhoeal agent, totally inhibited faecal proteinase activity in a ratio of 8:1 (weight:weight) diosmectite:enzyme protein. Mucolytic activity was assayed by release of degraded colonic mucin from the adherent mucus gel of freshly prepared pig colonic sacs in vitro. Trypsin (2 mgmlq) released a mean of 3.8 5:0.7 ~tg soluble muein per sac over 3.5 hours which was significantly three-fold greater than the control, mean 1.2 5:0.4 mg soluble mucin per sac. Diosmectite (100 mgml-~) effectively inhibited trypsin mucolytic activity with a mean of 0.4 5:0.2 nag degraded mu¢in produced per sac. This inhibitory action of diosmectito was shown to b e mediated by its (i) binding to trypsin and thereby inhibiting enzyme activity, and (ii) by binding to the adherent mucus layer (3.3 + 0.3/~g diosmectite per mm2 of mucosa).
These studies show (i) that several colonic mucolytic proteinase are raised in UC, (ii) there is a significant increase in negatively charged faecal proteinases in UC compared to controls, (iii) this activity is predominantly serine dependent proteiusses presumably from both the endogenous microflora and host enzyme secretions, (iv) diosmectite is an effective inhibitor of total colonic proteinase activity.
Immunology, Microbiology, and Inflammatory Disorders A909
O A DOUBLE-BLIND COMPARISON OF ORAL VERSUS RECTAL MESALAMINE VERSUS COMBINATION THERAPY IN THE TREATMENT OF DISTAL ULCERATIVE COLITIS. The Rowasa® Study Group: M. Safdi, M. DeMicco, C. Sninsky, P. Banks, L. Wruble, J. Deren, G. Koval, T. Nichols, S. Targan, D. Taylor, C. Fleishman, T. Nester. (Sites in OH, CA, FL, MA, TN, PA, OR). A 6-week, multieanter, randomized, double-blind trial was performed in 60 patients with active, histologically confirmed, mild to moderate distal ( ~ 50 era) ulcerative colitis (UC) to evaluate the efficacy and safety of oral (O) mesalamine tablets 800rag tid (n=22) vs. a 4g rectal (R) mesalamine enema qhs (n= 18) vs. the combination (C) of oral and rectal therapy (n=20). After baseline, patients were evaluated at weeks 1, 2, 3 and 6 with endoscopic evaluations at weeks 3 and 6. Efficacy evaluations included a disease activity index (DAD and a 7-point physician and patient clinical global impression (CGI) rating. The DAI represents a composite score of four subscales: evacuation frequency, rectal bleeding, mucosal appearance and physician's overall assessment of disease severity. Patient demographics, including UC history, did not differ significantly among treatment groups. Improvement in total DAI score was greater for C (-5.3 points) followed by R (-3.9) and O (-3.4) but this trend did not reach statistical significance. The subscale measuring rectal bleeding status was significantly different among treatments at weeks I, 2 and 6 (see table): Treatment % PatientsWithout Physician mean CGI scores revealed greater Period RectalBleeding improvement (p < 0.05) with C compared to O at all visits and R compared to O at weeks 2, 3 o R c and 6. At final assessment the percentage of Week 1 9 33* 40* patients rated by their physician as much or very much improved was 45, 78 and 89 for O, Week 2 lS 35 70* R and C, respectively. Greater improvement Week 3 43 5# 75 (P < 0.05) in patient CGI scores was observed for R compared to 0 and C compared to 0 at Week 6 36 56 95** weeks 2, 3 and 6. The percentage of patients * p
NEW METHOD FOR CHARACTERIZATION OF MUCIN IN INFLAMMATLY BOWEL DISEASE USING WHOLE GUT LAVAGE FLUID.
O SERUM BILE ACIDS, PLASMA CHOLECYSTOKININ (CCK), AND GALLBLADDER MOTILITY AFTER ILEAL RESECTION (IR). J.MJ.I. Salem~n~, F.M. Nagengast, A. van Schaik, A. Tangerman, W.P.M. Hopman, J.B.M.J. Jansen. Dept. of Gastroenterology, University Hospital Nijmegen, The Netherlands. IR is associated with bile acid malabsorption and an increased risk for cholelithiasis. A decreased duodenal bile acid output and increased biliary ursodeoxycholic acid (UDCA) fraction have been found after IR. The aim of this study was to examine whether IR leads to alterations in bile acid absorption, deconjugation, 7~-dehydroxylation, and formation of UDCA. Since intraduodenal bile acids may influence the release of CCK we further studied the effect of IR on plasma CCK and gallbladder volume (GBV). Methods: 8 patients with Crohn's disease and a history of IR (M/F=5/3, mean age 39, operation 1-14 yrs before this study, length of IR: 30-70 cm) and 12 healthy controls (M/F=8/4, mean age 43) were studied. None of the subjects were using cholestyramine. Fasting and postprandial blood samples were obtained at 15 rain intervals (3 hours) for conjugated (c) and unconjugated (unc) cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), and UDCA (gas-liquid chromatography) and CCK (RIA). GBV was measured using ultrasonography. Results: Controls IR Integrated serum bile acids (p.M. 180min) CA (c) 1854-26 83+32 p < 0.005 CDCA (c) 4544-67 2764-43 p <0.05 DCA (c) 194_+25 36_+5 p < 0.0001 UDCA (c) not detected not detected CA (unc) 44+6 974-23 p<0.05 CDCA (uric) 60+7 233_+51 p<0.01 DCA (unc) 65 _+9 56_+7 NS UDCA (unc) not detected 144+54 p<0.01 Fasting GBV (mL) 28 + 3 27 _+7 NS Percentage GB emptying (%) 78+ 3 83 +5 NS Fasting plasma CCK (pM) 3.4+0.2 3.54-0.4 NS Integrated CCK response (pM.180min) 898+73 961+ 130 NS Conclusions: postprandial conjugated bile acid levels are decreased while unconjuguted bile acid levels are increased after IR. Formation of DCA is reduced .after IR, probably as a result of rapid colonic transit with shortened exposure to bacterial 7~-dehydroxylase. The production of UDCA is increased after IR, which may be due to CDCA malabsorption. Bile acid malabsorption after IR does not lead to alterations in CCK release and gallbladder motility.
INC~ED SERINE DEPENDENT PROTEINASES IN ULCERATIVE COLITIS: MUCOLYSIS AND INHIBITION BY DIOSMECTITE H.J. Samson, J.P. Pearson, E.D. Srivastava, *M-T Droy-Lefalx and A. Allen, Department of Physiological Sciences, University of Newcastle upon Tyne, UK, and *Department of Pharmacology, Ipsen Institute, Paris, France.
H. Saitoh, Y. Uno, H. Nakajima, A. Munakata, M. Endo and Y. Yoshida. First Depts. of Internal Medichine & Biochemistry, Hirosaki University School of Medicine, Hirosaki, Japan Whole gut lavage fluid (WGLF) obtained using polyethyleneglycol-based orally administered balanced electrolyte solution prior to colonoscopy was examined in order to investigate the biochemical features of intestinal mucin in 20 normal subjects and 12 patients with inflammatory bowel disease (IBD) (5 with Crohn's disease (CD) and 7 with ulcerative colitis (UC)). Methods: Each WGLF sample was chromatographed on a gel-filtration column of Sepharose CL-4B, and then Sepharose CL-2B. Quantities of the mucin recovered in the void volume were then estimated. Ultracentrifugation was carried out to purify the mucin, followed by ion-exchange chromatography to separate neutral and acidic mucins. Results: In UC, the amount of mucin was decreased, whereas in CD, the amount of mucin was increased in comparison with normal subjects (p<0.05). Furthermore, the mucin acidity was estimated in terms of the acidic mucin/neutral mucin ratio (A/N). UC patients had a higher A/N than normal subjects (p<0.05).
subjects
l
[ normal
~
amount of mucin (mean±SD) mg/500 ml 14.99-18.13
(16.78)
10.s4-13.26 (11.971.
17.68-25.45 (20.89)*
A/N(mean±SD)
1.23-1.39(1.35)
2.87-3.69(3.2s)
i.Ii-1.42(i.14)
*p<0.05 vs. control Conclusion: The WGLF method is useful for investigation of mucin, and may reveal pathological changes in the mucosa in IBD.
Patients with ulcerative colitis (UC) have significantly raised total faecal proteinase activity compared to healthy controls. Using ion exchange HPLC faecal proteinases were ffactionated into two peaks of proteinase activity: pools I and 2. Pool 1 was eluted before application of a salt gradient and was coincident with 98% of pancreatic proteinase activity; pool 2 einted during the salt gradient. Mean proteinase activity (expressed in units of mmoles NI~ terminals min'lg"xdry weight of faeces, U) for pool 2 was significantly, six-fold higher for UC patients (6.8 5: 2.2U, n=9) than that for controls (1.2 5: 0.5U n = 10) p < 0.05. Mean proteinase activity for pool 1 from UC patients (4.6 5: 2.1U n = 9 ) was also six-fold greater than that for controls (0.7 5: 0.2U n = 10) although not significant. Specific inhibition showed that serine dependent proteinases were the major component ( > 76%) in pools 1 and 2 from both controls and UC patients. Diosmectito, a non-systemic, antidiarrhoeal agent, totally inhibited faecal proteinase activity in a ratio of 8:1 (weight:weight) diosmectite:enzyme protein. Mucolytic activity was assayed by release of degraded colonic mucin from the adherent mucus gel of freshly prepared pig colonic sacs in vitro. Trypsin (2 mgmlq) released a mean of 3.8 5:0.7 ~tg soluble muein per sac over 3.5 hours which was significantly three-fold greater than the control, mean 1.2 5:0.4 mg soluble mucin per sac. Diosmectite (100 mgml-~) effectively inhibited trypsin mucolytic activity with a mean of 0.4 5:0.2 nag degraded mu¢in produced per sac. This inhibitory action of diosmectito was shown to b e mediated by its (i) binding to trypsin and thereby inhibiting enzyme activity, and (ii) by binding to the adherent mucus layer (3.3 + 0.3/~g diosmectite per mm2 of mucosa).
These studies show (i) that several colonic mucolytic proteinase are raised in UC, (ii) there is a significant increase in negatively charged faecal proteinases in UC compared to controls, (iii) this activity is predominantly serine dependent proteiusses presumably from both the endogenous microflora and host enzyme secretions, (iv) diosmectite is an effective inhibitor of total colonic proteinase activity.