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13C labelled hiolein breath test as a non-invasive measurement of pancreatic exocrine function
A438 AGA ABSTRACTS
GASTROENTEROLOGYVol. 114, No, 4
• G1780 INFLUENCE OF GLUTATHIONE SYNTHESIS ON THE COURSE OF ACUTE EXPERIMENTAL PANCREATITIS IN THE RAT. G. Alsfasser, L. Herzog, M. Gock, J. Werner, E. Ryschich, J. Schmidt, M.M. Gebhard 1), Ch. Herfarth and E. Klar. Departments of Surgery and Experimental Surgery1), University of Heidelberg, Germany Introduction: Glutathione plays a central role as scavenger of reactive oxidative metabolites. L-Buthioninsulfoximine (BSO) is therefore, as inhibitor of "/-glutamylcystein-synthetase and by the way of glutathione synthesis, a potent amplifier of oxidative stress. We investigated the influence of BSO on the experimentally induced cerulein pancreatitis of the rat and on the cerulein stimulation of isolated pancreatic acini as correlate to oxidative stress. Methods: Male Wistar rats had a bolus of 1 ml 0,9% NaC1 with or wihtout (control) 8 pmol/kg KG BSO injected intravenously. After anesthesia with ketanest/pentobarbital, an acute pancreatitis was induced by time and pressure dependent intraductal infusion of 0,125 ml 10 mMol glucodesoxychollic acid (GDOC) and subsequent intravenous application of 5 pg/kg/h cerulein. After a maximum observation period of 24 hrs the rats were euthanasised and the pancreas removed for histological analysis. For preparation of isolated pancreatic acini, male Wistar rats had their pancreas removed and collagenase digested in a standardised fashion. Isolated acini were stimulated over 45 Minuten with declining concentrations of cerulein (10-7 to 10-12 M). Lipase and Amylase were measured in the supernatant. Results: L-Buthioninsulfoximin (BSO) significantly increased mortality in experimentally induced pancreatitis. 7 of 8 BSO animals died versus 0 of 6 controls (p=0,036, Mann-Whitney-U-Test). On cellular level, BSO reduced secretory activity of stimulated acini concerning amylase as well as lipase (p < 0,01 with each; paired t-test). Conclusion: BSO increases mortality in experimentally induced pancreatitis. This is probably due to early local and systemic decompensation glutathionedependent scavenging systems. On cellular level, this mechanism is probably valid through reduction of energy dependent transport under oxidative stress (cerulein). G1781 ELECTROPHYSIOLOGIC RESPONSE OF THE BOVINE PANCREATIC DUCT EPITIIEL1UNI TO ETHANOL EXPOSURE IN V/TRO. C. Alvarez, K. Huff, B.L. Bass. Dept. of Surgery, Univ. of Maryland and Baltimore VAMC, Baltimore, MD, 21201. The pathophysiology of alcoholic pancreatitis remains undefined, but heavy and prolonged used is generally considered essential. Here we used a novel method of pancreatic duct cell isolation to study the electrophysiological response of the duct epithelium to acute ethanol exposure. Methods: Segments of the bovine main pancreatic duct were harvested fresh and maintained in tissue culture for 72h to allow recovery from the dissection. The segments were then mounted on Ussing chambers bathed in modified Ringer's solution and exposed to 95%O2/5%CO2. We monitored transepithelial tissue resistance (R,, fffcm2), short-circuit current (Io, pA/cm2) and potential difference (PD, mV) while ethanol was added in varying concentrations to both the secretory and nutrient chambers. Results are reported as change over baseline (A). Responses within and between groups were compared with ANOVA. Results: The duct epithelium responds to levels of ethanol commensurate with legal drinking limits (0.1 g/dl) with a pattern typical of secretion, with the PD and I falling and the R~ remaining stable. At higher doses all parameters increase, suggestive of a secretory block, while at supraphysiological doses brief elevations in R,, I and PD are followed by steep declines. Control 0.1g/cfiEtOH 0.25g/cgEIOH 0.Sg/tflEtOH 5.0g/~EtOH n
5
4
6
6
4
15.0 g/d~EtOH 3
"nmt3mi~lOnfir 3rain lOmin 3rain lOlnin 3rain lOmin 3rain lOmin 3min lOmin ARt - - 1.00.30.2±0.24).8±0.51.3_+0.81.3_-¢0.82.8:~1.0 1.8±0.8~8.8±0.8~7.9+1.6"~ 7.0~:7.6 5.3±8.4 AI~ -- D.I:~).-2.6±1.6-8.9~3.53.4±1S4.5~9.5 3.3~1.: 2.4~8.7 2.3±4.3 -0~3±2.8 13±38 -21~49 APD -- D~2-+0.~-50~29 -75±25 67.'~33 67-+49 17±31 17±48 0-14) 75!-485~26.~-170(h:954" *: p < 0.01 VSControl; #: p < 0.05 vs baseline Conclusions: We demonstrated that duct cells can be obtained from the bovine main pancreatic duct, maintained in culture, and used for isolated electrophysiological study. These in vitro results agree with observations on the secretory response to ethanol on the intact pancreas and suggest a direct effect on duct cell secretory function without significant toxicity except at maximal doses. Chronic inhibition of ductal secretion could play a role in the pathogenesis of alcoholic pancreatitis.
• G1782 13C LABELLED HIOLEIN BREATH TEST AS A NON-INVASIVE MEASUREMENT OF PANCREATIC EXOCRINE FUNCTION. ST Amann. JG Herrera, L Somogyi, M Cintron, D Wagner 1, PP Toskes, University of Florida, Gainesville FL., 1Metabolic Solutions Inc, NH. There is a clear need for a simple, non-invasive test of pancreatic function with acceptable sensitivity and specificity. Our previous study with 13C hiolein (Martek BioSciences) and breath test technology suggested this fat substrate could detect pancreatic steatorrhea. 13C labeled hiolein (diffusely
labeled, 98% enriched) fat substrate was used in our current study of pancreatic exocrine function. Two large doses of fat loads were used to "stress" the invivo lipase activity in an attempt to improve separation of patient groups. Aims: To evaluate 1) 13C labeled hiolein (HBT) absorption, by CO2 breath test analysis, between healthy control and chronic pancreatitis (CP) (diagnosed by secretin test and clinical history) subjects. 2)The effect of a 60 gm and 100 gm fat load on substrate absorption in all groups of subjects. 3) Relationship of the HBT to invivo lipase activity and fecal fat. Methods: 7 subjects with chronic pancreatitis (CP) and 7 healthy controls (HC) underwent the following tests: 1) 72 hour fecal fat (FF) on a high fat diet (100 gm of fat/day), 2) HBT (substrate mixed in corn oil), each preceded by a 60 gm and 100 gm fat load in a randomly allocated sequence. HBT was dosed at 2 mg/kg and breath samples were obtained hourly from 6 to 12 hours post dose. Breath samples were analyzed (Metabolic Solutions, Inc) and reported as total %13C recovery in COr CP subjects had duodenal aspiration for lipase output after CCK analog stimulation. Results: The 7 CP subjects had a median(QI-Q3) FF of 10(7-15) gm/24 hr. Two of the CP subjects had normal FF(clinically moderate CP). The 7 HC subjects had a median(Q1-Q3) FF of 2.8(2.3-3.4) gm/24 hr, normal FF is < 7gm/24hr. The subjects were 64% female. Breath test results below: Total %
13CO2recovery / m e d i a n
Healthy Controls (HC) Chronic Panereatitis (CP)
(QI-Q3)
HBT + 60 gm 15.3 (12.6-18.6) 1.3 (1.1-3.3)
HBT + 100 gm 15.0 (9.8-19.7) 1.1 (0.8-11.1)
There was a curvilinear relationship between lipase output (KU/hr) and the fecal fat and % 13C hiolein recovery in CP subjects. The 100gm fat load had no effect on total recovery but delayed the time to peak recovery in 60%. Conclusions: The 13C labeled hiolein breath test with a 60 gm fat load clearly separated CP from controls, including 2 subjects with moderate CP, no steatorrhea but a diagnosis of CP by secretin testing. It appears that 13C hiolein and a 60 gm fat load can detect both moderate and severe CP subjects from controls. The 100 gm fat load with the HBT had little overlap, provided no improvement in separation of groups, but did delay peak %13C recovery. These data 1) demonstrate that impaired lipolysis in moderate CP may lead to an abnormal HBT in the absence of steatorrhea and 2) support the use of 13C hiolein with an optimal 60 gm fat load as a non-invasive breath test for both moderate and severe pancreatic exocrine insufficiency. D. Wagner is President and PP Toskes is a consultant of Metabolic Solutions. G1783 PANCREATIC STELLATE CELLS PRODUCE COLLAGEN AND OTHER EXTRACELLULAR MATRIX PROTEINS AND EXHIBIT A PROLIFERATIVE RESPONSE TO PLATELET-DERIVED GROWTH FACTOR (PDGF). M.V. Apte, P.S. Haber, C.S. Moran, G.W. McCaughan, *M.A. Korsten, R.C. Pirola, J.S. Wilson. Pancreatic Research Group, GI Unit, Prince of Wales Hospital, Sydney, Australia and *Mount Sinai School of Medicine, N.Y., N.Y. Pancreatic fibrosis is a central pathological feature of chronic pancreatitis. However, the pathogenesis of pancreatic fibrosis remains unknown. In the liver, increased synthesis and secretion of extracellular matrix proteins by activated hepatic stellate cells is the major mechanism responsible for the production of fibrosis. We have recently identified stellate cells in rat pancreas and have developed a method to isolate and culture these cells (Pancreas 15:425, 1997), which provides a novel in vitro model to study their biology. The aims of this study were to determine whether cultured pancreatic stellate ceils i) produce extracellular matrix (ECM) proteins and ii) demonstrate increased proliferation when incubated with platelet-derived growth factor (PDGF), an inflammatory cytokine known to stimulate the proliferation of hepatic stellate cells. Methods: Pancreatic stellate cells were isolated and cultured in Iscove Dulbecco's medium containing 10% fetal bovine serum and 4mM glutamine, in uncoated plastic wells. Stellate ceils were stained by immuuocytochemical techniques for the ECM proteins procollagen III, collagen I, laminin and fibronectin using specific polyclonal antibodies (ceils incubated with the relevant preimmune host serum served as negative controls). To assess the effect of PDGF on cell proliferation, cultured cells were passaged and replated at equal seeding densities. Duplicate wells were then incubated for 48 hours with increasing concentrations of PDGF and cell counts determined at the end of the incubation period. Cells incubated without PDGF served as controls. Results: iI lmmunocytochemistry for extracellular matrix proteins: Pancreatic stellate cells in culture stained strongly positive for all the ECM proteins tested - procollagen III, collagen I, fibronectin and laminin. 2) Effect of PDGF on cell proliferation: Incubation of stellate ceils with 1, 5 and 10 ng/ml PDGF resulted in a dose-dependent increase in the number of cells per well (no. of cells expressed as % of control: 164.1_+13.2, 226.6+_37.1 and 305.4+_57.0 respectively; n=4 separate cell preparations). Conclusions: Using immunocytochemistry, this study has shown that cultured pancreatic stellate cells have the capacity to produce collagen and other extracellular matrix proteins. In addition, we have shown that pancreatic stellate ceils exhibit increased proliferation in response to PDGF. Implication: The results of this study provide further evidence in support of an important role for pancreatic stellate cells in pancreatic fibmgenesis.
GASTROENTEROLOGYVol. 114, No, 4
• G1780 INFLUENCE OF GLUTATHIONE SYNTHESIS ON THE COURSE OF ACUTE EXPERIMENTAL PANCREATITIS IN THE RAT. G. Alsfasser, L. Herzog, M. Gock, J. Werner, E. Ryschich, J. Schmidt, M.M. Gebhard 1), Ch. Herfarth and E. Klar. Departments of Surgery and Experimental Surgery1), University of Heidelberg, Germany Introduction: Glutathione plays a central role as scavenger of reactive oxidative metabolites. L-Buthioninsulfoximine (BSO) is therefore, as inhibitor of "/-glutamylcystein-synthetase and by the way of glutathione synthesis, a potent amplifier of oxidative stress. We investigated the influence of BSO on the experimentally induced cerulein pancreatitis of the rat and on the cerulein stimulation of isolated pancreatic acini as correlate to oxidative stress. Methods: Male Wistar rats had a bolus of 1 ml 0,9% NaC1 with or wihtout (control) 8 pmol/kg KG BSO injected intravenously. After anesthesia with ketanest/pentobarbital, an acute pancreatitis was induced by time and pressure dependent intraductal infusion of 0,125 ml 10 mMol glucodesoxychollic acid (GDOC) and subsequent intravenous application of 5 pg/kg/h cerulein. After a maximum observation period of 24 hrs the rats were euthanasised and the pancreas removed for histological analysis. For preparation of isolated pancreatic acini, male Wistar rats had their pancreas removed and collagenase digested in a standardised fashion. Isolated acini were stimulated over 45 Minuten with declining concentrations of cerulein (10-7 to 10-12 M). Lipase and Amylase were measured in the supernatant. Results: L-Buthioninsulfoximin (BSO) significantly increased mortality in experimentally induced pancreatitis. 7 of 8 BSO animals died versus 0 of 6 controls (p=0,036, Mann-Whitney-U-Test). On cellular level, BSO reduced secretory activity of stimulated acini concerning amylase as well as lipase (p < 0,01 with each; paired t-test). Conclusion: BSO increases mortality in experimentally induced pancreatitis. This is probably due to early local and systemic decompensation glutathionedependent scavenging systems. On cellular level, this mechanism is probably valid through reduction of energy dependent transport under oxidative stress (cerulein). G1781 ELECTROPHYSIOLOGIC RESPONSE OF THE BOVINE PANCREATIC DUCT EPITIIEL1UNI TO ETHANOL EXPOSURE IN V/TRO. C. Alvarez, K. Huff, B.L. Bass. Dept. of Surgery, Univ. of Maryland and Baltimore VAMC, Baltimore, MD, 21201. The pathophysiology of alcoholic pancreatitis remains undefined, but heavy and prolonged used is generally considered essential. Here we used a novel method of pancreatic duct cell isolation to study the electrophysiological response of the duct epithelium to acute ethanol exposure. Methods: Segments of the bovine main pancreatic duct were harvested fresh and maintained in tissue culture for 72h to allow recovery from the dissection. The segments were then mounted on Ussing chambers bathed in modified Ringer's solution and exposed to 95%O2/5%CO2. We monitored transepithelial tissue resistance (R,, fffcm2), short-circuit current (Io, pA/cm2) and potential difference (PD, mV) while ethanol was added in varying concentrations to both the secretory and nutrient chambers. Results are reported as change over baseline (A). Responses within and between groups were compared with ANOVA. Results: The duct epithelium responds to levels of ethanol commensurate with legal drinking limits (0.1 g/dl) with a pattern typical of secretion, with the PD and I falling and the R~ remaining stable. At higher doses all parameters increase, suggestive of a secretory block, while at supraphysiological doses brief elevations in R,, I and PD are followed by steep declines. Control 0.1g/cfiEtOH 0.25g/cgEIOH 0.Sg/tflEtOH 5.0g/~EtOH n
5
4
6
6
4
15.0 g/d~EtOH 3
"nmt3mi~lOnfir 3rain lOmin 3rain lOlnin 3rain lOmin 3rain lOmin 3min lOmin ARt - - 1.00.30.2±0.24).8±0.51.3_+0.81.3_-¢0.82.8:~1.0 1.8±0.8~8.8±0.8~7.9+1.6"~ 7.0~:7.6 5.3±8.4 AI~ -- D.I:~).-2.6±1.6-8.9~3.53.4±1S4.5~9.5 3.3~1.: 2.4~8.7 2.3±4.3 -0~3±2.8 13±38 -21~49 APD -- D~2-+0.~-50~29 -75±25 67.'~33 67-+49 17±31 17±48 0-14) 75!-485~26.~-170(h:954" *: p < 0.01 VSControl; #: p < 0.05 vs baseline Conclusions: We demonstrated that duct cells can be obtained from the bovine main pancreatic duct, maintained in culture, and used for isolated electrophysiological study. These in vitro results agree with observations on the secretory response to ethanol on the intact pancreas and suggest a direct effect on duct cell secretory function without significant toxicity except at maximal doses. Chronic inhibition of ductal secretion could play a role in the pathogenesis of alcoholic pancreatitis.
• G1782 13C LABELLED HIOLEIN BREATH TEST AS A NON-INVASIVE MEASUREMENT OF PANCREATIC EXOCRINE FUNCTION. ST Amann. JG Herrera, L Somogyi, M Cintron, D Wagner 1, PP Toskes, University of Florida, Gainesville FL., 1Metabolic Solutions Inc, NH. There is a clear need for a simple, non-invasive test of pancreatic function with acceptable sensitivity and specificity. Our previous study with 13C hiolein (Martek BioSciences) and breath test technology suggested this fat substrate could detect pancreatic steatorrhea. 13C labeled hiolein (diffusely
labeled, 98% enriched) fat substrate was used in our current study of pancreatic exocrine function. Two large doses of fat loads were used to "stress" the invivo lipase activity in an attempt to improve separation of patient groups. Aims: To evaluate 1) 13C labeled hiolein (HBT) absorption, by CO2 breath test analysis, between healthy control and chronic pancreatitis (CP) (diagnosed by secretin test and clinical history) subjects. 2)The effect of a 60 gm and 100 gm fat load on substrate absorption in all groups of subjects. 3) Relationship of the HBT to invivo lipase activity and fecal fat. Methods: 7 subjects with chronic pancreatitis (CP) and 7 healthy controls (HC) underwent the following tests: 1) 72 hour fecal fat (FF) on a high fat diet (100 gm of fat/day), 2) HBT (substrate mixed in corn oil), each preceded by a 60 gm and 100 gm fat load in a randomly allocated sequence. HBT was dosed at 2 mg/kg and breath samples were obtained hourly from 6 to 12 hours post dose. Breath samples were analyzed (Metabolic Solutions, Inc) and reported as total %13C recovery in COr CP subjects had duodenal aspiration for lipase output after CCK analog stimulation. Results: The 7 CP subjects had a median(QI-Q3) FF of 10(7-15) gm/24 hr. Two of the CP subjects had normal FF(clinically moderate CP). The 7 HC subjects had a median(Q1-Q3) FF of 2.8(2.3-3.4) gm/24 hr, normal FF is < 7gm/24hr. The subjects were 64% female. Breath test results below: Total %
13CO2recovery / m e d i a n
Healthy Controls (HC) Chronic Panereatitis (CP)
(QI-Q3)
HBT + 60 gm 15.3 (12.6-18.6) 1.3 (1.1-3.3)
HBT + 100 gm 15.0 (9.8-19.7) 1.1 (0.8-11.1)
There was a curvilinear relationship between lipase output (KU/hr) and the fecal fat and % 13C hiolein recovery in CP subjects. The 100gm fat load had no effect on total recovery but delayed the time to peak recovery in 60%. Conclusions: The 13C labeled hiolein breath test with a 60 gm fat load clearly separated CP from controls, including 2 subjects with moderate CP, no steatorrhea but a diagnosis of CP by secretin testing. It appears that 13C hiolein and a 60 gm fat load can detect both moderate and severe CP subjects from controls. The 100 gm fat load with the HBT had little overlap, provided no improvement in separation of groups, but did delay peak %13C recovery. These data 1) demonstrate that impaired lipolysis in moderate CP may lead to an abnormal HBT in the absence of steatorrhea and 2) support the use of 13C hiolein with an optimal 60 gm fat load as a non-invasive breath test for both moderate and severe pancreatic exocrine insufficiency. D. Wagner is President and PP Toskes is a consultant of Metabolic Solutions. G1783 PANCREATIC STELLATE CELLS PRODUCE COLLAGEN AND OTHER EXTRACELLULAR MATRIX PROTEINS AND EXHIBIT A PROLIFERATIVE RESPONSE TO PLATELET-DERIVED GROWTH FACTOR (PDGF). M.V. Apte, P.S. Haber, C.S. Moran, G.W. McCaughan, *M.A. Korsten, R.C. Pirola, J.S. Wilson. Pancreatic Research Group, GI Unit, Prince of Wales Hospital, Sydney, Australia and *Mount Sinai School of Medicine, N.Y., N.Y. Pancreatic fibrosis is a central pathological feature of chronic pancreatitis. However, the pathogenesis of pancreatic fibrosis remains unknown. In the liver, increased synthesis and secretion of extracellular matrix proteins by activated hepatic stellate cells is the major mechanism responsible for the production of fibrosis. We have recently identified stellate cells in rat pancreas and have developed a method to isolate and culture these cells (Pancreas 15:425, 1997), which provides a novel in vitro model to study their biology. The aims of this study were to determine whether cultured pancreatic stellate ceils i) produce extracellular matrix (ECM) proteins and ii) demonstrate increased proliferation when incubated with platelet-derived growth factor (PDGF), an inflammatory cytokine known to stimulate the proliferation of hepatic stellate cells. Methods: Pancreatic stellate cells were isolated and cultured in Iscove Dulbecco's medium containing 10% fetal bovine serum and 4mM glutamine, in uncoated plastic wells. Stellate ceils were stained by immuuocytochemical techniques for the ECM proteins procollagen III, collagen I, laminin and fibronectin using specific polyclonal antibodies (ceils incubated with the relevant preimmune host serum served as negative controls). To assess the effect of PDGF on cell proliferation, cultured cells were passaged and replated at equal seeding densities. Duplicate wells were then incubated for 48 hours with increasing concentrations of PDGF and cell counts determined at the end of the incubation period. Cells incubated without PDGF served as controls. Results: iI lmmunocytochemistry for extracellular matrix proteins: Pancreatic stellate cells in culture stained strongly positive for all the ECM proteins tested - procollagen III, collagen I, fibronectin and laminin. 2) Effect of PDGF on cell proliferation: Incubation of stellate ceils with 1, 5 and 10 ng/ml PDGF resulted in a dose-dependent increase in the number of cells per well (no. of cells expressed as % of control: 164.1_+13.2, 226.6+_37.1 and 305.4+_57.0 respectively; n=4 separate cell preparations). Conclusions: Using immunocytochemistry, this study has shown that cultured pancreatic stellate cells have the capacity to produce collagen and other extracellular matrix proteins. In addition, we have shown that pancreatic stellate ceils exhibit increased proliferation in response to PDGF. Implication: The results of this study provide further evidence in support of an important role for pancreatic stellate cells in pancreatic fibmgenesis.