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This month in Gastroenterology
THIS MONTH
IN
GASTROENTEROLOGY
STUART J. SPECHLER AND EDITORS
Normal and Neoplastfc Tissues Separated by Fluorescence Spectroscopy n exposure to laser-generated ultraviolet radiation, certain tissues fluoresce, emitting light with wavelengths and intensities that can be measured by fluorescence spectroscopy. For some tissues, the fluorescence spectra induced by laser irradiation are sufficiently characteristic to distinguish normal from diseased specimens. Now, Kapadia et al. have studied the ability of laser-induced fluorescence spectroscopy to identify neoplastic colonic tissue. Fluorescence spectroscopy data obtained on 35 specimens of normal colonic mucosa and on 35 adenomatous polyps were subjected to multivariate linear regression analysis to estimate the wavelengths and intensities of fluorescent light that best separated normal and neoplastic mucosae. A formula derived from this regression analysis was used to calculate a laser-induced fluorescence (LIF) score, and the diagnostic accuracy of the LIF score in distinguishing neoplastic and nonneoplastic colonic tissue was verified in a separate prospective study. In the latter investigation, all of 34 normal colonic specimens and 15 of 16 hyperplastic polyps had LIF scores in the nonneoplastic range, whereas LIF scores were in the neoplastic range for all of 16 adenomatous polyps. These results suggest that laser-induced aorescence spectroscopy may be a useful test for differentiating neoplastic and nonneoplastic mucosae. Although this study was conducted in vitro, the fiberoptic system used could be adapted for perendoscopic use in vivo. If studies in vivo confirm the promising results of this investigation, then perendoscopic fluorescence spectroscopy might have substantial clinical utility. For example, endoscopic biopsy,forceps might be guided to areas that exhibit abnormal fluorescence spectra. Such abnormalities at the site of a polypectomy would suggest the presence of residual adenomatous tissue. Furthermore, fluorescence spectroscopy might immediately distinguish adenomatous polyps, which require excision, from hyperplastic polyps or pseudopolyps that need not be removed. . . . page 150
0
Antimitochondrial Antibodies in PBC Inhibit Mitochondrial Enzymes
M oat
of the energy necessary for human cells to function is generated in the mitochondria by the oxidation of fuel molecules through the citric acid cycle. Most fuel molecules enter this cycle in the form of ace@ coenzyme A (CoA). For example, glucose is metabolized in the cytosol to pyruvate that. in turn, is metabolized in the mitochondria to acetyl CoA. The oxidative decarboxylation of pyruvate to acetyl CoA is catalyzed by the pyruvate dehydrogenase (PDH) complex, an organized conglomerate of three major enzyme subunits designated El, E2, and E3. Patients with primary biliary cirrhosis usually have antimitochondrial antibodies (AMA), some of which have been shown to recognize antigenie determinants on the E2 subunit of PDH. Yoshida et al. have further characterized these mitochondrial antibodies. For all of 13 patients with primary biliary cirrhosis whose sera were positive for AMA by indirect immunofluorescence (a conventional clinical laboratory assay], sera also reacted with the E2 subunit of PDH. In contrast, sera from 23 patients with rheumatic diseases and from 30 patients with chronic active hepatitis, although all AMA positive by indirect immunofluorescence, did not recog nize PDH-EZ. Furthermore, purified immunoglobulin G from 12 of the 13 patients with primary biliaxy cirrhosis inhibited PDH activity with an intensity that correlated closely with the titer of antibody against PDH complex. These results suggest that AMAs in primary biliary cirrhosis can inhibit important mitochondrial enzyme systems. Further studies are required to determine whether such enzyme inhibition plays a pathogenetic role in this cholestatic liver disorder. . . . page 187
Refluxes Trigger Reflexes
P
erfusion of the esophagus with acid has been shown to cause reversible bronchoconstriction in patients with asthma, some of whom experience improved pulmonary function with. antireflux therapy. Wright et al. have
explored possible mechanisms for reflux-induced pulmonary abnormalities in a prospective investigation of 136 patients referred for esophageal manometry because of dysphagia, chest pain, or symptoms of gastroesophageal reflux disease. Perfusion of the esophagus with either O.IN hydrochloric acid or weakly acidic saline solution resulted in significant reductions in heart rate, airway flow (FEV,), and arterial oxygen saturation, whereas esophageal perfusion with sterile water had no apparent effects on these cardiopulmonary functions. For 18 patients, pretreatment with atropine appeared to blunt the cardiopulmonary effects of esophageal acid perfusion, suggesting that these effects are cholinergically mediated (perhaps through the vagi). These results suggest that acid in the esophagus can trigger reflexes that induce bronchoconstriction and changes in heart rate, even in subjects without apparent pulmonary disease. The clinical importance of this reflux reflex remains to be determined. . . . page 71
ib;;;;;l
Electrolyte Transport
M any
studies on electrolyte transport by the colon have used the Ussing chamber technique of measuring ion fluxes across an intact sheet of colonic epithelium. These investigations elucidate transmucosal electrolyte movements but provide little information on the transport properties of individual epithelial cell membranes. Using intracellular microelectrodes and other techniques, Sandle et al. have explored electrolyte transport pathways at the cellular level in surgically removed specimens of distal colon from patients with inflammatory bowel disease and from control subjects. Microelectrode studies showed that the decrease in transepithelial voltage characteristic of inflamed colonic mucosa was partly a manifestation of decreased voltage across the colonocyte’s basolateral membrane, the site of the electrogenic Na’ pump. The activity of this pump was investigated in colonic mucosa treated with nystatin, a [continued
on next page]
GASTROENTgRouxzY
1986$&1-2
THIS MONTH
IN
GASTROENTEROLOGY
polyene antibiotic that renders the apical membrane freely permeable to Na’. With the apical membrane offering little resistance to Na’ movement, any increase in short-circuit current induced by luminal Na+ is presumed to reflect the activity of the electrogenic Na + pump. Compared with uninflamed colonic mucosa, the maximal short-circuit current induced by Nat was decreased by 76% in the specimens with colitis, a phenomenon indicating markedly diminished activity of the basolateral Na’ pump. These findings suggest that electrophysiological abnormalities of colonic epithelial cell membranes might contribute to the diarrhea of inflammatory bowel disease. . page 97 Feed a Cold, Starve a Pancreas? ancreatic proteins appear to play a pathogenetic role in alcoholic pancreatitis. Morphological studies of the pancreas damaged by chronic alcohol ingestion typically reveal ducts obstructed by protein precipitates. High concentrations of protein are frequently found in the pancreatic juice of alcoholic subjects. Furthermore, acute injury may result from the activation of digestive enzymes, themselves proteins, within the pancreas. The pancreas depends on an adequate supply of dietary amino acids for normal protein synthesis, and some authorities have proposed that the risk of developing pancreatitis may be higher for the well-nourished alcoholic than for the alcoholic whose diet is deficient in protein. Korsten et al. have studied the effects of chronic ethanol ingestion on pancreatic protein synthesis and enzyme secretion in rats fed liquid diets that varied in protein content. Both protein synthesis and enzyme secretion were increased significantly by ethanol ingestion in rats consuming a protein-adequate diet; these stimula-
P
tory effects of ethanol were attenuated markedly in rats on a protein-deficient diet. Although good nutrition clearly is not a prerequisite for alcoholic pancreatitis, this study suggests that alcoholstimulated pancreatic protein synthesis and enzyme secretion (potentially damaging phenomena] may be increased by a diet adequate in protein content. . page 229 Mechanisms of Gakrin Release From Human G Cells fter binding to specific membrane receptors on an endocrine cell, a secretagogue can stimulate hormone release through one of two known mechanisms: (a) by activating adenylate cyclase, thereby increasing cellular cyclic adenosine monophosphate and activating the cyclic adenosine monophosphate-dependent protein kinases that regulate hormone release, or (b) by activating phospholipase C, which effects the hydrolysis of phosphatidylinositol, a phospholipid in the cell membrane, to diacylglycerol and inosito1 trisphosphate. Diacylglycerol activates protein kinase C, an enzyme involved in the regulation of hormone secretion, and phosphatidylinositol elevates cellular Ca’ that can activate other secretion-regulating protein kinases. Although a number of agents are known to stimulate gastrin release from antral and duodenal G cells in vivo, it is not clear whether these agents act directly on the G cells or indirectly by releasing other endocrine, paracrine, and neurocrine substances that secondarily stimulate gastrin release. To investigate gastrin secretion by G cells in a system that minimizes extraneous influences, Campos et al. studied the effects of various agents on cultured, G cell-enriched preparations of human antral mucosal cells. Forskolin, an activator of adenylate cyclase, stimulated gastrin secretion from the cultured G cells in a
A
(CONTINUED]
concentration-dependent fashion, as did the calcium ionophore A23187 (which increases intracellular Ca+) and fi-PMA, a phorbol ester that directly activates protein kinase C. These results suggest that gastrin release might be stimulated by activation of either of the two known mechanisms for stimulus-secretion coupling in the G cell. Furthermore, gastrin release in this system was stimulated by bombesin and inhibited by somatostatin, suggesting that these peptides exert their effects directly on the G cells. page 36
Enprostil May Potentiate Injury by Alcohol
Gastric
n animals, prostaglandins of the E series have been shown to protect the gastric mucosa from injury by diverse noxious agents including ethanol and boiling water. Indeed, misoprostil (a prostaglandin E, analogue) is used clinically to prevent gastric injury by aspirin and other nonsteroidal antiinflammatory drugs. Enprostil, a synthetic analogue of prostaglandin E,, has also been shown to limit aspirininduced gastric mucosal injury in humans. Therefore, enprostil should protect the stomach from injury by ethanol, right? Not so, according to Cohen et al. In a study of eight healthy men, the gross gastric injury induced by the perendoscopic application of 80% ethanol was increased significantly by pretreatment of the mucosa with enprostil. In fact, one subject was withdrawn from further study when intramucosal hemorrhages were seen to develop within 3 minutes of the topical application of enprostil alone. This unanticipated potentiation of ethanolinduced gastric injury requires confirmation. Pending confirmatory studies, nevertheless, it seems prudent to use prostaglandins cautiously in alcoholic patients. page 45
I
IN
GASTROENTEROLOGY
STUART J. SPECHLER AND EDITORS
Normal and Neoplastfc Tissues Separated by Fluorescence Spectroscopy n exposure to laser-generated ultraviolet radiation, certain tissues fluoresce, emitting light with wavelengths and intensities that can be measured by fluorescence spectroscopy. For some tissues, the fluorescence spectra induced by laser irradiation are sufficiently characteristic to distinguish normal from diseased specimens. Now, Kapadia et al. have studied the ability of laser-induced fluorescence spectroscopy to identify neoplastic colonic tissue. Fluorescence spectroscopy data obtained on 35 specimens of normal colonic mucosa and on 35 adenomatous polyps were subjected to multivariate linear regression analysis to estimate the wavelengths and intensities of fluorescent light that best separated normal and neoplastic mucosae. A formula derived from this regression analysis was used to calculate a laser-induced fluorescence (LIF) score, and the diagnostic accuracy of the LIF score in distinguishing neoplastic and nonneoplastic colonic tissue was verified in a separate prospective study. In the latter investigation, all of 34 normal colonic specimens and 15 of 16 hyperplastic polyps had LIF scores in the nonneoplastic range, whereas LIF scores were in the neoplastic range for all of 16 adenomatous polyps. These results suggest that laser-induced aorescence spectroscopy may be a useful test for differentiating neoplastic and nonneoplastic mucosae. Although this study was conducted in vitro, the fiberoptic system used could be adapted for perendoscopic use in vivo. If studies in vivo confirm the promising results of this investigation, then perendoscopic fluorescence spectroscopy might have substantial clinical utility. For example, endoscopic biopsy,forceps might be guided to areas that exhibit abnormal fluorescence spectra. Such abnormalities at the site of a polypectomy would suggest the presence of residual adenomatous tissue. Furthermore, fluorescence spectroscopy might immediately distinguish adenomatous polyps, which require excision, from hyperplastic polyps or pseudopolyps that need not be removed. . . . page 150
0
Antimitochondrial Antibodies in PBC Inhibit Mitochondrial Enzymes
M oat
of the energy necessary for human cells to function is generated in the mitochondria by the oxidation of fuel molecules through the citric acid cycle. Most fuel molecules enter this cycle in the form of ace@ coenzyme A (CoA). For example, glucose is metabolized in the cytosol to pyruvate that. in turn, is metabolized in the mitochondria to acetyl CoA. The oxidative decarboxylation of pyruvate to acetyl CoA is catalyzed by the pyruvate dehydrogenase (PDH) complex, an organized conglomerate of three major enzyme subunits designated El, E2, and E3. Patients with primary biliary cirrhosis usually have antimitochondrial antibodies (AMA), some of which have been shown to recognize antigenie determinants on the E2 subunit of PDH. Yoshida et al. have further characterized these mitochondrial antibodies. For all of 13 patients with primary biliary cirrhosis whose sera were positive for AMA by indirect immunofluorescence (a conventional clinical laboratory assay], sera also reacted with the E2 subunit of PDH. In contrast, sera from 23 patients with rheumatic diseases and from 30 patients with chronic active hepatitis, although all AMA positive by indirect immunofluorescence, did not recog nize PDH-EZ. Furthermore, purified immunoglobulin G from 12 of the 13 patients with primary biliaxy cirrhosis inhibited PDH activity with an intensity that correlated closely with the titer of antibody against PDH complex. These results suggest that AMAs in primary biliary cirrhosis can inhibit important mitochondrial enzyme systems. Further studies are required to determine whether such enzyme inhibition plays a pathogenetic role in this cholestatic liver disorder. . . . page 187
Refluxes Trigger Reflexes
P
erfusion of the esophagus with acid has been shown to cause reversible bronchoconstriction in patients with asthma, some of whom experience improved pulmonary function with. antireflux therapy. Wright et al. have
explored possible mechanisms for reflux-induced pulmonary abnormalities in a prospective investigation of 136 patients referred for esophageal manometry because of dysphagia, chest pain, or symptoms of gastroesophageal reflux disease. Perfusion of the esophagus with either O.IN hydrochloric acid or weakly acidic saline solution resulted in significant reductions in heart rate, airway flow (FEV,), and arterial oxygen saturation, whereas esophageal perfusion with sterile water had no apparent effects on these cardiopulmonary functions. For 18 patients, pretreatment with atropine appeared to blunt the cardiopulmonary effects of esophageal acid perfusion, suggesting that these effects are cholinergically mediated (perhaps through the vagi). These results suggest that acid in the esophagus can trigger reflexes that induce bronchoconstriction and changes in heart rate, even in subjects without apparent pulmonary disease. The clinical importance of this reflux reflex remains to be determined. . . . page 71
ib;;;;;l
Electrolyte Transport
M any
studies on electrolyte transport by the colon have used the Ussing chamber technique of measuring ion fluxes across an intact sheet of colonic epithelium. These investigations elucidate transmucosal electrolyte movements but provide little information on the transport properties of individual epithelial cell membranes. Using intracellular microelectrodes and other techniques, Sandle et al. have explored electrolyte transport pathways at the cellular level in surgically removed specimens of distal colon from patients with inflammatory bowel disease and from control subjects. Microelectrode studies showed that the decrease in transepithelial voltage characteristic of inflamed colonic mucosa was partly a manifestation of decreased voltage across the colonocyte’s basolateral membrane, the site of the electrogenic Na’ pump. The activity of this pump was investigated in colonic mucosa treated with nystatin, a [continued
on next page]
GASTROENTgRouxzY
1986$&1-2
THIS MONTH
IN
GASTROENTEROLOGY
polyene antibiotic that renders the apical membrane freely permeable to Na’. With the apical membrane offering little resistance to Na’ movement, any increase in short-circuit current induced by luminal Na+ is presumed to reflect the activity of the electrogenic Na + pump. Compared with uninflamed colonic mucosa, the maximal short-circuit current induced by Nat was decreased by 76% in the specimens with colitis, a phenomenon indicating markedly diminished activity of the basolateral Na’ pump. These findings suggest that electrophysiological abnormalities of colonic epithelial cell membranes might contribute to the diarrhea of inflammatory bowel disease. . page 97 Feed a Cold, Starve a Pancreas? ancreatic proteins appear to play a pathogenetic role in alcoholic pancreatitis. Morphological studies of the pancreas damaged by chronic alcohol ingestion typically reveal ducts obstructed by protein precipitates. High concentrations of protein are frequently found in the pancreatic juice of alcoholic subjects. Furthermore, acute injury may result from the activation of digestive enzymes, themselves proteins, within the pancreas. The pancreas depends on an adequate supply of dietary amino acids for normal protein synthesis, and some authorities have proposed that the risk of developing pancreatitis may be higher for the well-nourished alcoholic than for the alcoholic whose diet is deficient in protein. Korsten et al. have studied the effects of chronic ethanol ingestion on pancreatic protein synthesis and enzyme secretion in rats fed liquid diets that varied in protein content. Both protein synthesis and enzyme secretion were increased significantly by ethanol ingestion in rats consuming a protein-adequate diet; these stimula-
P
tory effects of ethanol were attenuated markedly in rats on a protein-deficient diet. Although good nutrition clearly is not a prerequisite for alcoholic pancreatitis, this study suggests that alcoholstimulated pancreatic protein synthesis and enzyme secretion (potentially damaging phenomena] may be increased by a diet adequate in protein content. . page 229 Mechanisms of Gakrin Release From Human G Cells fter binding to specific membrane receptors on an endocrine cell, a secretagogue can stimulate hormone release through one of two known mechanisms: (a) by activating adenylate cyclase, thereby increasing cellular cyclic adenosine monophosphate and activating the cyclic adenosine monophosphate-dependent protein kinases that regulate hormone release, or (b) by activating phospholipase C, which effects the hydrolysis of phosphatidylinositol, a phospholipid in the cell membrane, to diacylglycerol and inosito1 trisphosphate. Diacylglycerol activates protein kinase C, an enzyme involved in the regulation of hormone secretion, and phosphatidylinositol elevates cellular Ca’ that can activate other secretion-regulating protein kinases. Although a number of agents are known to stimulate gastrin release from antral and duodenal G cells in vivo, it is not clear whether these agents act directly on the G cells or indirectly by releasing other endocrine, paracrine, and neurocrine substances that secondarily stimulate gastrin release. To investigate gastrin secretion by G cells in a system that minimizes extraneous influences, Campos et al. studied the effects of various agents on cultured, G cell-enriched preparations of human antral mucosal cells. Forskolin, an activator of adenylate cyclase, stimulated gastrin secretion from the cultured G cells in a
A
(CONTINUED]
concentration-dependent fashion, as did the calcium ionophore A23187 (which increases intracellular Ca+) and fi-PMA, a phorbol ester that directly activates protein kinase C. These results suggest that gastrin release might be stimulated by activation of either of the two known mechanisms for stimulus-secretion coupling in the G cell. Furthermore, gastrin release in this system was stimulated by bombesin and inhibited by somatostatin, suggesting that these peptides exert their effects directly on the G cells. page 36
Enprostil May Potentiate Injury by Alcohol
Gastric
n animals, prostaglandins of the E series have been shown to protect the gastric mucosa from injury by diverse noxious agents including ethanol and boiling water. Indeed, misoprostil (a prostaglandin E, analogue) is used clinically to prevent gastric injury by aspirin and other nonsteroidal antiinflammatory drugs. Enprostil, a synthetic analogue of prostaglandin E,, has also been shown to limit aspirininduced gastric mucosal injury in humans. Therefore, enprostil should protect the stomach from injury by ethanol, right? Not so, according to Cohen et al. In a study of eight healthy men, the gross gastric injury induced by the perendoscopic application of 80% ethanol was increased significantly by pretreatment of the mucosa with enprostil. In fact, one subject was withdrawn from further study when intramucosal hemorrhages were seen to develop within 3 minutes of the topical application of enprostil alone. This unanticipated potentiation of ethanolinduced gastric injury requires confirmation. Pending confirmatory studies, nevertheless, it seems prudent to use prostaglandins cautiously in alcoholic patients. page 45
I