GASTROENTEROLOGY
1992;103:1367-1371
CORRESPONDENCE Readers are encouraged to write Letters to the Editor concerning articles that have been published in GASTROENTEROLOGY. Short, general comments are also considered, but use of the Correspondence section for publication of original data in preliminary form is not encouraged. Letters should be typewritten double-spaced and submitted in triplicate.
Septicemia After Endoscopic Retrograde Cholangiopancreatography Dear Sir: Risk factors for septicemia following endoscopic retrograde cholangiopancreatography (ERCP) with biliary stenting have been reported by Motte et al. in a retrospective study including 347 patients.’ According to these investigators the main predictive factor for septicemia was the poor quality of biliary drainage following endoscopy. The most frequently found species, Pseudomonas aeruginosa, was significantly associated with formerly performed ERCP in other centers. In our experience, we found 51 cases of septicemia among 2010 patients (2.5%). Septicemia was defined as the positivity of at least two blood cultures within 72 hours after ERCP (blood cultures were not obtained systematically after endoscopic procedures but only in cases with suspected infections). Our study did not include patients who had developed septicemia, cholangitis, fever, or extrabiliary infections within 48 hours before ERCP. The patients who had developed septicemia after ERCP were compared with those in a control group of noninfected patients (n = 193). These control patients were selected at random (one every 10 patients) among all the patients who had undergone ERCP during the same period. The average number of endoscopic or percutaneous transhepatic procedures performed was 1.76+ 1.12in the infected group and 1.25+ 0.70in the control group (P = 0.001). In the infected group, it was the first endoscopic procedure in 28 cases (55%) the second in 14 cases (27%),and the third or more in 9 cases (18%). The prevalence of malignant biliary stricture was higher in the group of infected patients than in the control patients (80% vs. 23%; P = 0.0001). In the infected group, biliary stenting had been performed in 35 cases. Biliary drainage was considered complete in 16 cases (46%) and incomplete in 19 cases (54%). Our septicemic rate following biliary stenting (8.9%) was comparable with the figure reported by Motte et al.’ (10%). In both univariate and multivariate analysis, we have looked for infectious risk factors following ERCP: incomplete biliary drainage (P = 0.009) and number of ERCP sessions (P = 0.001) were found to be predictive factors for infection, in agreement with these authors. Moreover, malignancy (P = 0.0001) as well as ultrasound dilatation of the biliary tract (P < 0.001)were also significant predictive factors. P. aeruginosa was also the most frequently found species (accounting for 80% of positive blood cultures and 23% of positive bile cultures), but, unlike these authors, its prevalence was not higher in patients having formerly undergone recent ERCP in other centers. Therefore, in agreement with Motte et al.,‘.z we believe that if malignancy is suspected, antibiotic prophylaxis with anti-P. aeruginosa activity should be administered before ERCP. If biliary drainage is incomplete after a first ERCP, this antibiotherapy should be continued until obstruction is relieved. This antibiotic prophylaxis is likely to reduce the rate of septicemia following
ERCP in patients with malignant drainage is difficult to obtain.
strictures
in whom complete
P. NOVELLO, M.D. H. HAGEGE, M.D. C. BUFFET,PH.D. J.FRITSCH,M.D. A. CHOURY, M.D. J.P.ETIENNE,PH.D. Service des Maladies du Foie et de I’AppareiI Digestif, Hopital de Bicdtre Kremlin Bicdtre, France 1. Motte S, Deviere J, Dumonceau JM, Serruys E, Thys JP, Cremer M. Risk factors for septicemia following endoscopic biliary stenting. Gastroenterology 1991;101:1374-1381. 2. Deviere J, Motte S, Dumonceau JM, Serruys E, Thys JP, Cremer M. Septicemia after endoscopic retrograde cholangiopancreatography. Endoscopy 1990;22:72-75.
Quantitative Discrimination B and C Receptors
of Glomerular
Dear Sir: Morgan et al. recently investigated binding of atria1 natriuretic factor (ANF) on freshly isolated glomeruli of bile duct-ligated (BDL) rats without ascites and sham-operated controls.’ They found a single ANF binding site with an affinity of about 5 X lo-” mol/L in both BDL rats and controls with a higher receptor density in the BDL rats (1221+249vs.931 f 209 fmol/mgprotein). At the same time they found no difference in cyclic guanosine monophosphate (GMP) generation of isolated glomeruli between BDL rats and controls. They conclude that their “findings of increased density of total receptors with no change in guanylate cyclasecontaining receptors suggest that the density of C receptors is increased in glomeruli from cirrhotic rats.” The binding sites found by the authors had an affinity of about 5 X lo-" mol/L, well in the range that we have previously reported for glomerular C receptors. Unfortunately, they did not characterize the nature of their binding site, e.g., by affinity crosslinking and sodium dodecyl sulfate gel electrophoresis. Following ANF administration, the authors observed cyclic GMP production by glomeruli, suggesting the existence of B receptors. Why were they unable to demonstrate B receptors in their binding studies? Referring to our previous work,2 Morgan et al. suggest that differences in methodology should be responsible, namely their using freshly isolated intact glomeruli whereas our experiments were performed on frozen glomerular membranes. At the time of the submission of Morgan’s paper, we had published a detailed investigation on optimization of glomerular ANF binding studies.3 At various durations and temperatures of incubation we had compared binding of ANF to intact glomeruli as opposed to frozen membranes. We were able to determine quantitatively both B and C receptors in glomeruli as well as in glomerular membranes. However, interassay variations were lower and specific binding was higher with the membrane preparation than with the glomer-
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GASTROENTEROLOGY Vol. 103,No. 4
uli. These results suggested that glomerular membrane preparations are at least as suitable as intact glomeruli for the purpose of ANF binding studies. Morgan et al. tried to back up their binding studies by quoting Ballermann et a1.,4who found only one type of ANF receptor on intact glomeruli. It seems worth mentioning that this study was published in 1985, 2 years before Maack et al5 detected a second type of ANF receptor using a synthetic ANF analogue. A more recent work by Ballermann’s group’ showed both ANF receptors on intact glomeruli. Morgan et al. furthermore state that their acid wash of glomeruli at pH 5 might have contributed to their inability of detecting a second ANF binding site. In our hands, acid wash at pH 5, however, does not affect the detection of both types of ANF receptors.’ Indeed, the most likely explanation for the authors’ failure to demonstrate a second ANF binding site seems to be problems with their methodology. In our binding-displacement studies we used 15 different ANF concentrations in a concentration range of 10~‘2-10-6 mol/L, with a significant number of concentrations in the lower range. This is particularly important in order to not miss the B receptor with higher affinity than the C receptor (in the 5 x lo-” mol/L range). Morgan et al., however, while stating in their Materials and Methods that they used eight different concentrations in the 10-“-10-8 mol/L range, show in Figure 1 of their paper six different concentrations, but in the 10-‘“-10-6 mol/L range. Thus, they do not provide any data in the low ANF concentration range important for the detection of the B receptor. Morgan et al. state a lack of information about regulation of ANF receptors in health and disease, quoting a study on increased ANF-receptor density in rats following sodium restriction or water deprivation8 Indeed, after various experimental procedures, a blunted biological response and reduced cyclic GMP generation had been observed despite an increase of total glomerular ANF-receptor density.4,g In 1990 we were able to offer an explanation for this seeming contradiction by showing that the doubling of ANF-receptor density following water deprivation in rats is due to an exclusive increase of C receptors with no change of B receptors.” Thus, it seems that changes of the density of the C receptor involved in the clearance of ANF rather than of the guanylate cyclase-coupled B receptor are important in physiological and pathophysiological regulations of the ANF system. Altogether, investigations of the glomerular ANF-receptor status can and should comprise quantitative discrimination of both ANF receptor types. ALEXANDER L.GERBES,M.D. Klinikum Grojhadern Ludwig-Maximilians-Universitot Postfach 7oL 260 5000Miinchen 70,Germany
Miinchen
1. Morgan TR, Morgan K, Jonas GM, Thillainadarajah
I. Atria1 cirrhosis in rats. Gastroen-
natriuretic factor in experimental terology 1992;102:1356-1362. 2. Gerbes AL, Kollenda MC, Vollmar AM, Reichen J, Vakil N, Scarborough RM. Density of two types of glomerular ANF receptors is altered in BDL rats with ascites. Hepatology 1991;13:562-566. 3. Kollenda MC, Scarborough RM, Gerbes AL. Discrimination and quantification of glomerular receptor subtypes for atria1 natriuretic factor (ANF). J Receptor Res 1991;11:259-273. 4. Ballermann BJ, Hoover L, Karnovsky MJ, Brenner BM. Physiologic regulation of atria1 natriuretic factor receptors in rat renal glomeruli. J Clin Invest 1985;76:2049-2056, 5. Maack T, Suzuki M, Almeida FA, Nussenzveig D, Scarborough RM, McEnroe GA, Lewicki JA. Physiological role of silent receptors of atria1 natriuretic factor. Science 1987; 238:675-678.
6. Martin ER, Lewicki JA, Scarborough RM, Ballermann BJ. Expression and regulation of ANP receptor subtypes in rat renal glomeruli and papillae. Am J Physiol 1989:257:F649-F657. 7. Kollenda M. Dissertationsschrift der Ludwigs-MaximiliansUniversitat Mtinchen, Munich, Germany, vorgelegt 1991. 8. Gauquelin G, Garcia R, Carrier F, Cantin M, Gutkowska J, Thibault G, Schiffrin EL. Glomerular ANF receptor regulation during changes in sodium and water metabolism. Am J Physiol 1988;254:F51-F55. 9. Cachofeiro V, Schiffrin EL, Cantin M, Garcia R. Glomerular and vascular atria1 natriuretic factor receptors in adrenalectomized rats. Am J Physiol 1989;256:R1250-1257. 10. Kollenda MC, Vollmar AM, McEnroe GA, Gerbes AL. Dehydration increases the density of C-receptors for ANF on rat glomerular membranes. Am J Physiol1990;258:R1084-R1088. Reply. We appreciate Dr. Gerbes’ letter regarding our recent study of ANF in cirrhotic rats.’ As we noted in our article Dr. Gerbes and colleagues had published studies of glomerular ANF receptors in ascitic rats,*but we were unaware of his study of ANF receptors in glomerular membranes and in intact glomeruli at the time of submission of our manuscript3 Our glomerular receptor binding studies were performed using eight concentrations of ANF over the range of 1O-“-1O-8 mol/L. Subsequently, we performed glomerular receptor binding studies in portal vein-ligated and sham rats over the range of 10-‘*-10-8 mol/L ANF.4 We used three concentrations of ANF over each log change in ANF concentration. We performed this second set of receptor binding studies over an extended range and with three points per log in order to not miss a second binding site with a high affinity. We were unable to find two binding sites (by LIGAND analysis] in any of our sham or portal vein-ligated rats (more than 30 experiments). We believe two or more ANF receptors are present in glomeruli from our sham and BDL rats. However, we were unable to show two binding sites using our system. We did not perform affinity cross-linking studies or experiments with C-ANF to look more closely for the C receptor. Much of the information about ANF receptors has been reviewed by Martin and Ballermann.’ Most of the studies of ANF receptors have used receptor binding studies alone. Of the 66 studies of ANF receptors cited in this review, only 4 found two receptors in their receptor binding studies. Thus, our results are similar to the findings of many other investigators. As stated, differences in methodology may explain why some investigators find two receptors in their receptor binding studies and others find only one. Another explanation for finding only one receptor in the receptor binding studies is that the B and C receptors may have the same affinity. TIMOTHY R. MORGAN, M.D. KENGATHEVY MORGAN, PH.D. University of California, Irvine-Long Beach Medical Program Veterans Administration Medical Center 5901 East Seventh Street Long Beach, California 90822 Morgan TR, Morgan K, Jonas G, Thillainadarajah I. Atria1 natriuretic factor in experimental cirrhosis in rats. Gastroenterology 1992;102:1356-1362. Gerbes AL, Kollenda MC, Vollmar AM, Reichen J, Vakil N, Scarborough RM. Density of two types of glomerular ANF receptors is altered in BDL rats with ascites. Hepatology 1991;13:562-566. Kollenda MC, Scarborough RM, Gerbes AI. Discrimination and quantification of glomerular receptor subtypes for atria1 natriuretic factor. J Receptor Res 1991;11:259-73.
1992;103:1367-1371
CORRESPONDENCE Readers are encouraged to write Letters to the Editor concerning articles that have been published in GASTROENTEROLOGY. Short, general comments are also considered, but use of the Correspondence section for publication of original data in preliminary form is not encouraged. Letters should be typewritten double-spaced and submitted in triplicate.
Septicemia After Endoscopic Retrograde Cholangiopancreatography Dear Sir: Risk factors for septicemia following endoscopic retrograde cholangiopancreatography (ERCP) with biliary stenting have been reported by Motte et al. in a retrospective study including 347 patients.’ According to these investigators the main predictive factor for septicemia was the poor quality of biliary drainage following endoscopy. The most frequently found species, Pseudomonas aeruginosa, was significantly associated with formerly performed ERCP in other centers. In our experience, we found 51 cases of septicemia among 2010 patients (2.5%). Septicemia was defined as the positivity of at least two blood cultures within 72 hours after ERCP (blood cultures were not obtained systematically after endoscopic procedures but only in cases with suspected infections). Our study did not include patients who had developed septicemia, cholangitis, fever, or extrabiliary infections within 48 hours before ERCP. The patients who had developed septicemia after ERCP were compared with those in a control group of noninfected patients (n = 193). These control patients were selected at random (one every 10 patients) among all the patients who had undergone ERCP during the same period. The average number of endoscopic or percutaneous transhepatic procedures performed was 1.76+ 1.12in the infected group and 1.25+ 0.70in the control group (P = 0.001). In the infected group, it was the first endoscopic procedure in 28 cases (55%) the second in 14 cases (27%),and the third or more in 9 cases (18%). The prevalence of malignant biliary stricture was higher in the group of infected patients than in the control patients (80% vs. 23%; P = 0.0001). In the infected group, biliary stenting had been performed in 35 cases. Biliary drainage was considered complete in 16 cases (46%) and incomplete in 19 cases (54%). Our septicemic rate following biliary stenting (8.9%) was comparable with the figure reported by Motte et al.’ (10%). In both univariate and multivariate analysis, we have looked for infectious risk factors following ERCP: incomplete biliary drainage (P = 0.009) and number of ERCP sessions (P = 0.001) were found to be predictive factors for infection, in agreement with these authors. Moreover, malignancy (P = 0.0001) as well as ultrasound dilatation of the biliary tract (P < 0.001)were also significant predictive factors. P. aeruginosa was also the most frequently found species (accounting for 80% of positive blood cultures and 23% of positive bile cultures), but, unlike these authors, its prevalence was not higher in patients having formerly undergone recent ERCP in other centers. Therefore, in agreement with Motte et al.,‘.z we believe that if malignancy is suspected, antibiotic prophylaxis with anti-P. aeruginosa activity should be administered before ERCP. If biliary drainage is incomplete after a first ERCP, this antibiotherapy should be continued until obstruction is relieved. This antibiotic prophylaxis is likely to reduce the rate of septicemia following
ERCP in patients with malignant drainage is difficult to obtain.
strictures
in whom complete
P. NOVELLO, M.D. H. HAGEGE, M.D. C. BUFFET,PH.D. J.FRITSCH,M.D. A. CHOURY, M.D. J.P.ETIENNE,PH.D. Service des Maladies du Foie et de I’AppareiI Digestif, Hopital de Bicdtre Kremlin Bicdtre, France 1. Motte S, Deviere J, Dumonceau JM, Serruys E, Thys JP, Cremer M. Risk factors for septicemia following endoscopic biliary stenting. Gastroenterology 1991;101:1374-1381. 2. Deviere J, Motte S, Dumonceau JM, Serruys E, Thys JP, Cremer M. Septicemia after endoscopic retrograde cholangiopancreatography. Endoscopy 1990;22:72-75.
Quantitative Discrimination B and C Receptors
of Glomerular
Dear Sir: Morgan et al. recently investigated binding of atria1 natriuretic factor (ANF) on freshly isolated glomeruli of bile duct-ligated (BDL) rats without ascites and sham-operated controls.’ They found a single ANF binding site with an affinity of about 5 X lo-” mol/L in both BDL rats and controls with a higher receptor density in the BDL rats (1221+249vs.931 f 209 fmol/mgprotein). At the same time they found no difference in cyclic guanosine monophosphate (GMP) generation of isolated glomeruli between BDL rats and controls. They conclude that their “findings of increased density of total receptors with no change in guanylate cyclasecontaining receptors suggest that the density of C receptors is increased in glomeruli from cirrhotic rats.” The binding sites found by the authors had an affinity of about 5 X lo-" mol/L, well in the range that we have previously reported for glomerular C receptors. Unfortunately, they did not characterize the nature of their binding site, e.g., by affinity crosslinking and sodium dodecyl sulfate gel electrophoresis. Following ANF administration, the authors observed cyclic GMP production by glomeruli, suggesting the existence of B receptors. Why were they unable to demonstrate B receptors in their binding studies? Referring to our previous work,2 Morgan et al. suggest that differences in methodology should be responsible, namely their using freshly isolated intact glomeruli whereas our experiments were performed on frozen glomerular membranes. At the time of the submission of Morgan’s paper, we had published a detailed investigation on optimization of glomerular ANF binding studies.3 At various durations and temperatures of incubation we had compared binding of ANF to intact glomeruli as opposed to frozen membranes. We were able to determine quantitatively both B and C receptors in glomeruli as well as in glomerular membranes. However, interassay variations were lower and specific binding was higher with the membrane preparation than with the glomer-
1368 CORRESPONDENCE
GASTROENTEROLOGY Vol. 103,No. 4
uli. These results suggested that glomerular membrane preparations are at least as suitable as intact glomeruli for the purpose of ANF binding studies. Morgan et al. tried to back up their binding studies by quoting Ballermann et a1.,4who found only one type of ANF receptor on intact glomeruli. It seems worth mentioning that this study was published in 1985, 2 years before Maack et al5 detected a second type of ANF receptor using a synthetic ANF analogue. A more recent work by Ballermann’s group’ showed both ANF receptors on intact glomeruli. Morgan et al. furthermore state that their acid wash of glomeruli at pH 5 might have contributed to their inability of detecting a second ANF binding site. In our hands, acid wash at pH 5, however, does not affect the detection of both types of ANF receptors.’ Indeed, the most likely explanation for the authors’ failure to demonstrate a second ANF binding site seems to be problems with their methodology. In our binding-displacement studies we used 15 different ANF concentrations in a concentration range of 10~‘2-10-6 mol/L, with a significant number of concentrations in the lower range. This is particularly important in order to not miss the B receptor with higher affinity than the C receptor (in the 5 x lo-” mol/L range). Morgan et al., however, while stating in their Materials and Methods that they used eight different concentrations in the 10-“-10-8 mol/L range, show in Figure 1 of their paper six different concentrations, but in the 10-‘“-10-6 mol/L range. Thus, they do not provide any data in the low ANF concentration range important for the detection of the B receptor. Morgan et al. state a lack of information about regulation of ANF receptors in health and disease, quoting a study on increased ANF-receptor density in rats following sodium restriction or water deprivation8 Indeed, after various experimental procedures, a blunted biological response and reduced cyclic GMP generation had been observed despite an increase of total glomerular ANF-receptor density.4,g In 1990 we were able to offer an explanation for this seeming contradiction by showing that the doubling of ANF-receptor density following water deprivation in rats is due to an exclusive increase of C receptors with no change of B receptors.” Thus, it seems that changes of the density of the C receptor involved in the clearance of ANF rather than of the guanylate cyclase-coupled B receptor are important in physiological and pathophysiological regulations of the ANF system. Altogether, investigations of the glomerular ANF-receptor status can and should comprise quantitative discrimination of both ANF receptor types. ALEXANDER L.GERBES,M.D. Klinikum Grojhadern Ludwig-Maximilians-Universitot Postfach 7oL 260 5000Miinchen 70,Germany
Miinchen
1. Morgan TR, Morgan K, Jonas GM, Thillainadarajah
I. Atria1 cirrhosis in rats. Gastroen-
natriuretic factor in experimental terology 1992;102:1356-1362. 2. Gerbes AL, Kollenda MC, Vollmar AM, Reichen J, Vakil N, Scarborough RM. Density of two types of glomerular ANF receptors is altered in BDL rats with ascites. Hepatology 1991;13:562-566. 3. Kollenda MC, Scarborough RM, Gerbes AL. Discrimination and quantification of glomerular receptor subtypes for atria1 natriuretic factor (ANF). J Receptor Res 1991;11:259-273. 4. Ballermann BJ, Hoover L, Karnovsky MJ, Brenner BM. Physiologic regulation of atria1 natriuretic factor receptors in rat renal glomeruli. J Clin Invest 1985;76:2049-2056, 5. Maack T, Suzuki M, Almeida FA, Nussenzveig D, Scarborough RM, McEnroe GA, Lewicki JA. Physiological role of silent receptors of atria1 natriuretic factor. Science 1987; 238:675-678.
6. Martin ER, Lewicki JA, Scarborough RM, Ballermann BJ. Expression and regulation of ANP receptor subtypes in rat renal glomeruli and papillae. Am J Physiol 1989:257:F649-F657. 7. Kollenda M. Dissertationsschrift der Ludwigs-MaximiliansUniversitat Mtinchen, Munich, Germany, vorgelegt 1991. 8. Gauquelin G, Garcia R, Carrier F, Cantin M, Gutkowska J, Thibault G, Schiffrin EL. Glomerular ANF receptor regulation during changes in sodium and water metabolism. Am J Physiol 1988;254:F51-F55. 9. Cachofeiro V, Schiffrin EL, Cantin M, Garcia R. Glomerular and vascular atria1 natriuretic factor receptors in adrenalectomized rats. Am J Physiol 1989;256:R1250-1257. 10. Kollenda MC, Vollmar AM, McEnroe GA, Gerbes AL. Dehydration increases the density of C-receptors for ANF on rat glomerular membranes. Am J Physiol1990;258:R1084-R1088. Reply. We appreciate Dr. Gerbes’ letter regarding our recent study of ANF in cirrhotic rats.’ As we noted in our article Dr. Gerbes and colleagues had published studies of glomerular ANF receptors in ascitic rats,*but we were unaware of his study of ANF receptors in glomerular membranes and in intact glomeruli at the time of submission of our manuscript3 Our glomerular receptor binding studies were performed using eight concentrations of ANF over the range of 1O-“-1O-8 mol/L. Subsequently, we performed glomerular receptor binding studies in portal vein-ligated and sham rats over the range of 10-‘*-10-8 mol/L ANF.4 We used three concentrations of ANF over each log change in ANF concentration. We performed this second set of receptor binding studies over an extended range and with three points per log in order to not miss a second binding site with a high affinity. We were unable to find two binding sites (by LIGAND analysis] in any of our sham or portal vein-ligated rats (more than 30 experiments). We believe two or more ANF receptors are present in glomeruli from our sham and BDL rats. However, we were unable to show two binding sites using our system. We did not perform affinity cross-linking studies or experiments with C-ANF to look more closely for the C receptor. Much of the information about ANF receptors has been reviewed by Martin and Ballermann.’ Most of the studies of ANF receptors have used receptor binding studies alone. Of the 66 studies of ANF receptors cited in this review, only 4 found two receptors in their receptor binding studies. Thus, our results are similar to the findings of many other investigators. As stated, differences in methodology may explain why some investigators find two receptors in their receptor binding studies and others find only one. Another explanation for finding only one receptor in the receptor binding studies is that the B and C receptors may have the same affinity. TIMOTHY R. MORGAN, M.D. KENGATHEVY MORGAN, PH.D. University of California, Irvine-Long Beach Medical Program Veterans Administration Medical Center 5901 East Seventh Street Long Beach, California 90822 Morgan TR, Morgan K, Jonas G, Thillainadarajah I. Atria1 natriuretic factor in experimental cirrhosis in rats. Gastroenterology 1992;102:1356-1362. Gerbes AL, Kollenda MC, Vollmar AM, Reichen J, Vakil N, Scarborough RM. Density of two types of glomerular ANF receptors is altered in BDL rats with ascites. Hepatology 1991;13:562-566. Kollenda MC, Scarborough RM, Gerbes AI. Discrimination and quantification of glomerular receptor subtypes for atria1 natriuretic factor. J Receptor Res 1991;11:259-73.
