- Email: [email protected]
The bilirubin controversy: Settled?
488
SELECTED SUMMARIES
consideration has been the role of medications used during the prodromal illness. This pilot project was planned originally to evaluate the feasibility and methods to be used in a larger study to characterize the relationship of Reye’s syndrome to medication use. The findings in the initial study strongly suggest an association between the use of aspirin during a prodromal illness and the subsequent development of Reye’s syndrome. Because of these striking observations, a committee of the Institute of Medicine, National Academy of Sciences, which advised the Public Health Service Reye Syndrome Task Force, recommended publication of this preliminary report while awaiting the findings of the larger study. The observations in this study agree with previous reports of the use of salicylates immediately before the onset of Reye’s syndrome in as many as 90%-109% of children with this disorder, as compared with 49%-70% of the controls [Pediatrics 1980; 66:859-64, JAMA 1982;247:3089-94, JAMA 1982;248:687-91). However, several potential problems were identified in previous reports and have been addressed in the present study, including the possibility of “differential recall” among the parents of case and control patients, inclusion of patients who do not have Reye’s syndrome, and failure to include all patients with Reye’s syndrome at a center. A prospective study of children presenting with only vomiting and minimal elevations of aminotransferase levels suggests that mild Reye’s syndrome (grade I) occurs more commonly than previously appreciated (N Engl J Med 1983;309:133-9). The incidence of Reye’s syndrome (biopsy-proven cases of grade I or greater] was estimated in that study to be a minimum of 3.5 cases per 100,000 children per year in an urban United States population. About 80% of the patients with grade I Reye’s syndrome had taken salicylates before the onset of the disorder. The present study suggests at least as great a use of salicylates among a carefully evaluated group of patients with grade II or greater encephalopathy. Considering both groups, it appears that Reye’s syndrome associated with salicylate use may be more common than previously realized. However, the association of salicylates with grade I Reye’s syndrome may not have great clinical importance because most of these patients do not progress to more severe neurologic involvement. A recent extension of this association is the description of the development of Reye’s syndrome in children receiving chronic salicylate therapy for connective tissue disease (Pediatrics 1985; 76202-5, J Pediatr 1985;107:877-80). In the Public Health Service report, 1 patient received salicylate treatment for juvenile rheumatoid arthritis, another for Kawasaki’s syndrome. The presentation of Reye’s syndrome in this group appears to be distinct from the syndrome of salicylate hepatotoxicity in patients with connective tissue disease (Arthritis Rheum 1973;16:1-9, Ann Intern Med 1974;80:1-8, J Pediatr 1978;93:1034-7). Salicylate hepatotoxicity in patients with connective tissue disease is dose-related, and additionally, the histopathology in most of the described patients is distinct from that of Reye’s syndrome. Furthermore, rechallenge with salicylates has caused recurrence of liver dysfunction in the former group. Liver failure and encephalopathy can develop with salicylate hepatotoxicity, as in Reye’s syndrome (J Pediatr 1978;93:1034-7). No prospective study is available to ascertain whether the risk of Reye’s syndrome is increased in patients undergoing chronic salicylate therapy as compared with children who receive intermittent salicylate therapy for acute illnesses. In recent years, there has been a massive campaign to alert the public about the potential risk of salicylates in the etiology of Reye’s syndrome. Federal regulations requiring warnings on aspirin labels have been approved by the United States Department of Health and Human Services to begin in 1986. The incidence of
GASTROENTEROLOGY Vol. 91, No. 2
Reye’s syndrome has been decreasing steadily across the nation (MMWR 1984;33:41-2). A concomitant decrease in the use of salicylates in children and the occurrence of Reye’s syndrome over the past 5 yr has been described in a report from Michigan (Pediatrics 1986;77:93-8). The authors are careful not to ascribe a definite causal relationship; however, they suggest that this observation adds further support to the hypothesis that aspirin is an etiologic factor in this syndrome. The case-control study resulting from this pilot project could provide the final evidence necessary to establish a cause and effect relationship. However, Reye’s syndrome fortunately seems to be disappearing, possibly as a result of decreased use of aspirin, and the final study may never be done. M. H. ULSHEN, M.D.
THE BILIRUBIN CONTROVERSY: SETTLED? Peters WHM, Jansen PLA4 (Division of Gastrointestinal and HosLiver Diseases, Department of Medicine, University pital Nijmegen, Nijmegen, The Netherlands) Microsomal UDP-glucuronyltransferase-catalyzed bilirubin diglucuronide formation in human liver. J Hepatol 1986;2:182-94 (March). The catalytic properties of human liver microsomal bilirubin UDP-glucuronyltransferase toward unconjugated bilirubin and bilirubin monoglucuronide were determined on microsomal preparations and on liver homogenates. Conjugation of bilirubin was performed on SDS-activated microsomes. Under the experimental conditions used, human microsomes catalyzed the formation both of bilirubin monoglucuronide from unconjugated bilirubin and of bilirubin diglucuronide from monoglucuronide. The pH optima was 7.8 for the former reaction, and 7.4 for the latter. To answer the key question, whether the enzyme can form bilirubin diglucuronide in the amounts found in human bile, unconjugated bilirubin was incubated with UDP-glucuronic acid in various concentrations. At 4 mM UDP-glucuronic acid, unconjugated bilirubin (at IO-12 PM) was almost entirely conjugated with formation of 80% diglucuronide and 18% monoglucuronide. This was in the range of values determined on human bile specimens. Based on these results, the total capacity of human liver microsomes to form bilirubin diglucuronide was estimated: it was 3-3.6 mmohday, i.e., about 10 times normal daily bilirubin diglucuronide excretion in humans. Finally, by radiation-inactivation analysis, the authors estimated that the molecular weight of the enzyme catalyzing monoglucuronide formation was -55,000, whereas that catalyzing diglucuronide formation was -209,000. The authors imply that, in vivo, in humans, bilirubin diglucuronide formation is probably entirely catalyzed by microsomal UDP-glucuronyltransferase. They suggest that monoglucuronide formation is catalyzed by the monomeric form of the enzyme, with a molecular weight of 55,000, whereas diglucuronide formation is catalyzed by a tetramer with a molecular weight of 209,000. Comment. After the discovery, in the late 1956s, that “directreacting” bilirubin was in fact a water-soluble diglucuronide, an
SELECTED SUMMARIES
August 1986
active, search for the enzyme responsible for transforming bilirubin into bilirubin glucuronide began. It was quickly established that bilirubin glucuronide was formed in the endoplasmic reticulum; however, with the microsomal preparations used, only monoglucuronide was formed. Although conventional teaching and most textbooks indicated that bilirubin was conjugated in the endoplasmic reticulum, there was no clear evidence that the diglucuronide was formed there. In 1977, Jansen et al. proposed that diglucuronide formation from monoglucuronide was catalyzed by an enzyme located in the hepatocyte plasma membrane (J Biol Chem 1977;252:2710-6). The proposed enzyme catalyzed the formation of one molecule of diglucuronide from two molecules of monoglucuronide, with liberation of one molecule of unconjugated bilirubin (transmutation or transglucuronidation). This theory was supported by the observation that the intrahepatic pool of conjugated bilirubin appeared to be almost exclusively monoglucuronide (J Clin Invest 1978:61:142-9). These findings greatly stimulated research in this area, but soon generated much controversy, mostly between the west and east coasts of the United States. In brief, this view was challenged by two types of evidence: other workers were unable to find this activity in rat liver plasma membranes (J Biol Chem 1984;259:5500-6, J Biol Chem 1984;259:4527-33), and the transmutation reaction was found to be the result of nonenzymatic dipyrole exchange (J Clin Invest 1982;69:3547-57). In addition, experiments in normal and Gunn rats in vivo supported the view that bilirubin diglucuronide was actually formed by the UDP-glucuronyltransferase system (J Clin Invest 1980;65:1332-42), and diglucuronide formation by rat liver microsomes was also demonstrated (Proc Nat1 Acad Sci USA 1979;76:2037-41, Can J Biochem 1980;58:130210). This paper seems to definitely settle the controversy and extends the previous findings to humans. Peters and Jansen elegantly demonstrate that human liver microsomes, in vitro, catalyze the conjugation of bilirubin up to the diglucuronide. Microsomes are able to form conjugates in the proportion found in normal human bile. In addition, the finding of appreciable amounts of diglucuronide in the microsomes themselves makes it very unlikely that diglucuronide is formed at the plasma membrane. The calculation of total liver capacity for diglucuronide formation suggests that the normal liver has a considerable overcapacity (about lo-fold). This may explain why some patients with Gilbert’s syndrome and a >85% reduction in conjugating capacity have a modestly elevated serum unconjugated bilirubin and only a slight reduction in biliary diglucuronide to monoglucuronide ratio. Such controversies are frequently strong stimulants for research in a given area. It is hoped that this brief overview and account of this final settlement will be useful for those readers who have not followed the story throughout. S. ERLINGER, M.D.
FLEXIBLE SIGMOIDOSCOPY IS SOME ENOUGH?
TRAINING-
Bowman MA, Wherry DC [Georgetown University School of Medicine, Washington, DC.) Training for flexible sigmoidoscopy. Gastrointest Endsoc 1985;31:309-12 (October) .
A survey was conducted among 326 participants of l-day flexible sigmoidoscopy programs. These courses consisted of didactic sessions, as well as intubation into plastic colon models and anesthetized dogs. Of the 144 (44%) individuals responding, 20% had some prior expe-
489
rience with flexible sigmoidoscopy. About one-half of the responders (75 physicians) with no previous endoscopic experience reported using a 3O- or 60-cm flexible sigmoidoscope after the course. Of these, one-third obtained additional training after the course. Overall, about 30% of those presently performing flexible sigmoidoscopy had prior or subsequent training. About 20% of the responders were not doing flexible sigmoidoscopy after the course. Responders felt that the instrument was easy to use. Most examiners, however, reported using both the 60-cm flexible and the rigid sigmoidoscope together in their practice, and about three-quarters of the responders continued to use the rigid sigmoidoscope 6 mo after the course. One perforation of the colon was reported during an attempt at polypectomy despite the didactic recommendations against polypectomy during flexible sigmoidoscopy. The authors concluded that the “one-day course was successful in preparing the majority of responders in the use of the flexible sigmoidoscope”. Comment. The advantages of the flexible (30 or 60 cm) over the rigid sigmoidoscope are well documented (Dis Colon Rectum 1979;22:162-8, Gastrointest Endosc 1982;28:233-6) and the replacement of the rigid with the flexible sigmoidoscope for nearly all indications is advocated. Indeed, if a significant impact on the mortality of colorectal cancer is to be made, it is likely to require large scale utilization of these instruments by primary care physicians. Practicing nonendoscopic physicians have been slow to switch over to flexible sigmoidoscopy and increase its utilization for screening, as is advocated by the American Cancer Society (CA 1983;23:368-9). There are many reasons for this lethargy, including difficulty in obtaining training or experience with the flexible instrument sufficient to provide competency in its use. In addition, it is difficult in a busy practice to commit time to mastering the technique, and there is perceived a considerable expense in purchasing the system (Gastrointest Endosc 1983;29:186a). For privilege granting for the performance of flexible sigmoidoscopy in the hospital, a demonstration of competency is a minimal standard in most institutions, as is recommended for the practice of all endoscopic procedures (Hosp Med Staff 1982; 11:2-5). It is not felt that short courses, including the utilization of plastic models, will provide this competency. Hands-on experience in patients is required before competency will be obtained. Variations in the number of cases required to reach competency have been reported. One well-constructed study (Gastrointest Endosc 1984;30:1043a) indicated that a minimum of 15 cases for individuals already faculative for rigid sigmoidoscopy is necessary. In fact, the American Society for Gastrointestinal Endoscopy and the American Academy of Family Physicians, in recognition of this necessity, have constructed a national one-on-one preceptor training program of no less than 10 cases for 30-cm flexible sigmoidoscopy and 15 cases for 60-cm flexible sigmoidoscopy. The latter number is still likely to fall short of providing full competency for many trainees but may serve as a satisfactory background for additional experience. Unmentioned, however, in most of the discussions concerning training is the development of skills other than mechanical dexterity. It is vital that the training encompass not only depth of insertion, but also pathology recognition and understanding of appropriate subsequent management. Indeed, evidence shows that there is little to be gained by longer (60 cm] intubation over a 30-cm examination (Gastrointest Endosc 1984;30:59-64). One-on-one preceptor courses have the advantages of providing an interpretation of pathology and important opportunities for discussions regarding management of colorectal conditions. The roles of biopsy, polypectomy, follow-ups, and
SELECTED SUMMARIES
consideration has been the role of medications used during the prodromal illness. This pilot project was planned originally to evaluate the feasibility and methods to be used in a larger study to characterize the relationship of Reye’s syndrome to medication use. The findings in the initial study strongly suggest an association between the use of aspirin during a prodromal illness and the subsequent development of Reye’s syndrome. Because of these striking observations, a committee of the Institute of Medicine, National Academy of Sciences, which advised the Public Health Service Reye Syndrome Task Force, recommended publication of this preliminary report while awaiting the findings of the larger study. The observations in this study agree with previous reports of the use of salicylates immediately before the onset of Reye’s syndrome in as many as 90%-109% of children with this disorder, as compared with 49%-70% of the controls [Pediatrics 1980; 66:859-64, JAMA 1982;247:3089-94, JAMA 1982;248:687-91). However, several potential problems were identified in previous reports and have been addressed in the present study, including the possibility of “differential recall” among the parents of case and control patients, inclusion of patients who do not have Reye’s syndrome, and failure to include all patients with Reye’s syndrome at a center. A prospective study of children presenting with only vomiting and minimal elevations of aminotransferase levels suggests that mild Reye’s syndrome (grade I) occurs more commonly than previously appreciated (N Engl J Med 1983;309:133-9). The incidence of Reye’s syndrome (biopsy-proven cases of grade I or greater] was estimated in that study to be a minimum of 3.5 cases per 100,000 children per year in an urban United States population. About 80% of the patients with grade I Reye’s syndrome had taken salicylates before the onset of the disorder. The present study suggests at least as great a use of salicylates among a carefully evaluated group of patients with grade II or greater encephalopathy. Considering both groups, it appears that Reye’s syndrome associated with salicylate use may be more common than previously realized. However, the association of salicylates with grade I Reye’s syndrome may not have great clinical importance because most of these patients do not progress to more severe neurologic involvement. A recent extension of this association is the description of the development of Reye’s syndrome in children receiving chronic salicylate therapy for connective tissue disease (Pediatrics 1985; 76202-5, J Pediatr 1985;107:877-80). In the Public Health Service report, 1 patient received salicylate treatment for juvenile rheumatoid arthritis, another for Kawasaki’s syndrome. The presentation of Reye’s syndrome in this group appears to be distinct from the syndrome of salicylate hepatotoxicity in patients with connective tissue disease (Arthritis Rheum 1973;16:1-9, Ann Intern Med 1974;80:1-8, J Pediatr 1978;93:1034-7). Salicylate hepatotoxicity in patients with connective tissue disease is dose-related, and additionally, the histopathology in most of the described patients is distinct from that of Reye’s syndrome. Furthermore, rechallenge with salicylates has caused recurrence of liver dysfunction in the former group. Liver failure and encephalopathy can develop with salicylate hepatotoxicity, as in Reye’s syndrome (J Pediatr 1978;93:1034-7). No prospective study is available to ascertain whether the risk of Reye’s syndrome is increased in patients undergoing chronic salicylate therapy as compared with children who receive intermittent salicylate therapy for acute illnesses. In recent years, there has been a massive campaign to alert the public about the potential risk of salicylates in the etiology of Reye’s syndrome. Federal regulations requiring warnings on aspirin labels have been approved by the United States Department of Health and Human Services to begin in 1986. The incidence of
GASTROENTEROLOGY Vol. 91, No. 2
Reye’s syndrome has been decreasing steadily across the nation (MMWR 1984;33:41-2). A concomitant decrease in the use of salicylates in children and the occurrence of Reye’s syndrome over the past 5 yr has been described in a report from Michigan (Pediatrics 1986;77:93-8). The authors are careful not to ascribe a definite causal relationship; however, they suggest that this observation adds further support to the hypothesis that aspirin is an etiologic factor in this syndrome. The case-control study resulting from this pilot project could provide the final evidence necessary to establish a cause and effect relationship. However, Reye’s syndrome fortunately seems to be disappearing, possibly as a result of decreased use of aspirin, and the final study may never be done. M. H. ULSHEN, M.D.
THE BILIRUBIN CONTROVERSY: SETTLED? Peters WHM, Jansen PLA4 (Division of Gastrointestinal and HosLiver Diseases, Department of Medicine, University pital Nijmegen, Nijmegen, The Netherlands) Microsomal UDP-glucuronyltransferase-catalyzed bilirubin diglucuronide formation in human liver. J Hepatol 1986;2:182-94 (March). The catalytic properties of human liver microsomal bilirubin UDP-glucuronyltransferase toward unconjugated bilirubin and bilirubin monoglucuronide were determined on microsomal preparations and on liver homogenates. Conjugation of bilirubin was performed on SDS-activated microsomes. Under the experimental conditions used, human microsomes catalyzed the formation both of bilirubin monoglucuronide from unconjugated bilirubin and of bilirubin diglucuronide from monoglucuronide. The pH optima was 7.8 for the former reaction, and 7.4 for the latter. To answer the key question, whether the enzyme can form bilirubin diglucuronide in the amounts found in human bile, unconjugated bilirubin was incubated with UDP-glucuronic acid in various concentrations. At 4 mM UDP-glucuronic acid, unconjugated bilirubin (at IO-12 PM) was almost entirely conjugated with formation of 80% diglucuronide and 18% monoglucuronide. This was in the range of values determined on human bile specimens. Based on these results, the total capacity of human liver microsomes to form bilirubin diglucuronide was estimated: it was 3-3.6 mmohday, i.e., about 10 times normal daily bilirubin diglucuronide excretion in humans. Finally, by radiation-inactivation analysis, the authors estimated that the molecular weight of the enzyme catalyzing monoglucuronide formation was -55,000, whereas that catalyzing diglucuronide formation was -209,000. The authors imply that, in vivo, in humans, bilirubin diglucuronide formation is probably entirely catalyzed by microsomal UDP-glucuronyltransferase. They suggest that monoglucuronide formation is catalyzed by the monomeric form of the enzyme, with a molecular weight of 55,000, whereas diglucuronide formation is catalyzed by a tetramer with a molecular weight of 209,000. Comment. After the discovery, in the late 1956s, that “directreacting” bilirubin was in fact a water-soluble diglucuronide, an
SELECTED SUMMARIES
August 1986
active, search for the enzyme responsible for transforming bilirubin into bilirubin glucuronide began. It was quickly established that bilirubin glucuronide was formed in the endoplasmic reticulum; however, with the microsomal preparations used, only monoglucuronide was formed. Although conventional teaching and most textbooks indicated that bilirubin was conjugated in the endoplasmic reticulum, there was no clear evidence that the diglucuronide was formed there. In 1977, Jansen et al. proposed that diglucuronide formation from monoglucuronide was catalyzed by an enzyme located in the hepatocyte plasma membrane (J Biol Chem 1977;252:2710-6). The proposed enzyme catalyzed the formation of one molecule of diglucuronide from two molecules of monoglucuronide, with liberation of one molecule of unconjugated bilirubin (transmutation or transglucuronidation). This theory was supported by the observation that the intrahepatic pool of conjugated bilirubin appeared to be almost exclusively monoglucuronide (J Clin Invest 1978:61:142-9). These findings greatly stimulated research in this area, but soon generated much controversy, mostly between the west and east coasts of the United States. In brief, this view was challenged by two types of evidence: other workers were unable to find this activity in rat liver plasma membranes (J Biol Chem 1984;259:5500-6, J Biol Chem 1984;259:4527-33), and the transmutation reaction was found to be the result of nonenzymatic dipyrole exchange (J Clin Invest 1982;69:3547-57). In addition, experiments in normal and Gunn rats in vivo supported the view that bilirubin diglucuronide was actually formed by the UDP-glucuronyltransferase system (J Clin Invest 1980;65:1332-42), and diglucuronide formation by rat liver microsomes was also demonstrated (Proc Nat1 Acad Sci USA 1979;76:2037-41, Can J Biochem 1980;58:130210). This paper seems to definitely settle the controversy and extends the previous findings to humans. Peters and Jansen elegantly demonstrate that human liver microsomes, in vitro, catalyze the conjugation of bilirubin up to the diglucuronide. Microsomes are able to form conjugates in the proportion found in normal human bile. In addition, the finding of appreciable amounts of diglucuronide in the microsomes themselves makes it very unlikely that diglucuronide is formed at the plasma membrane. The calculation of total liver capacity for diglucuronide formation suggests that the normal liver has a considerable overcapacity (about lo-fold). This may explain why some patients with Gilbert’s syndrome and a >85% reduction in conjugating capacity have a modestly elevated serum unconjugated bilirubin and only a slight reduction in biliary diglucuronide to monoglucuronide ratio. Such controversies are frequently strong stimulants for research in a given area. It is hoped that this brief overview and account of this final settlement will be useful for those readers who have not followed the story throughout. S. ERLINGER, M.D.
FLEXIBLE SIGMOIDOSCOPY IS SOME ENOUGH?
TRAINING-
Bowman MA, Wherry DC [Georgetown University School of Medicine, Washington, DC.) Training for flexible sigmoidoscopy. Gastrointest Endsoc 1985;31:309-12 (October) .
A survey was conducted among 326 participants of l-day flexible sigmoidoscopy programs. These courses consisted of didactic sessions, as well as intubation into plastic colon models and anesthetized dogs. Of the 144 (44%) individuals responding, 20% had some prior expe-
489
rience with flexible sigmoidoscopy. About one-half of the responders (75 physicians) with no previous endoscopic experience reported using a 3O- or 60-cm flexible sigmoidoscope after the course. Of these, one-third obtained additional training after the course. Overall, about 30% of those presently performing flexible sigmoidoscopy had prior or subsequent training. About 20% of the responders were not doing flexible sigmoidoscopy after the course. Responders felt that the instrument was easy to use. Most examiners, however, reported using both the 60-cm flexible and the rigid sigmoidoscope together in their practice, and about three-quarters of the responders continued to use the rigid sigmoidoscope 6 mo after the course. One perforation of the colon was reported during an attempt at polypectomy despite the didactic recommendations against polypectomy during flexible sigmoidoscopy. The authors concluded that the “one-day course was successful in preparing the majority of responders in the use of the flexible sigmoidoscope”. Comment. The advantages of the flexible (30 or 60 cm) over the rigid sigmoidoscope are well documented (Dis Colon Rectum 1979;22:162-8, Gastrointest Endosc 1982;28:233-6) and the replacement of the rigid with the flexible sigmoidoscope for nearly all indications is advocated. Indeed, if a significant impact on the mortality of colorectal cancer is to be made, it is likely to require large scale utilization of these instruments by primary care physicians. Practicing nonendoscopic physicians have been slow to switch over to flexible sigmoidoscopy and increase its utilization for screening, as is advocated by the American Cancer Society (CA 1983;23:368-9). There are many reasons for this lethargy, including difficulty in obtaining training or experience with the flexible instrument sufficient to provide competency in its use. In addition, it is difficult in a busy practice to commit time to mastering the technique, and there is perceived a considerable expense in purchasing the system (Gastrointest Endosc 1983;29:186a). For privilege granting for the performance of flexible sigmoidoscopy in the hospital, a demonstration of competency is a minimal standard in most institutions, as is recommended for the practice of all endoscopic procedures (Hosp Med Staff 1982; 11:2-5). It is not felt that short courses, including the utilization of plastic models, will provide this competency. Hands-on experience in patients is required before competency will be obtained. Variations in the number of cases required to reach competency have been reported. One well-constructed study (Gastrointest Endosc 1984;30:1043a) indicated that a minimum of 15 cases for individuals already faculative for rigid sigmoidoscopy is necessary. In fact, the American Society for Gastrointestinal Endoscopy and the American Academy of Family Physicians, in recognition of this necessity, have constructed a national one-on-one preceptor training program of no less than 10 cases for 30-cm flexible sigmoidoscopy and 15 cases for 60-cm flexible sigmoidoscopy. The latter number is still likely to fall short of providing full competency for many trainees but may serve as a satisfactory background for additional experience. Unmentioned, however, in most of the discussions concerning training is the development of skills other than mechanical dexterity. It is vital that the training encompass not only depth of insertion, but also pathology recognition and understanding of appropriate subsequent management. Indeed, evidence shows that there is little to be gained by longer (60 cm] intubation over a 30-cm examination (Gastrointest Endosc 1984;30:59-64). One-on-one preceptor courses have the advantages of providing an interpretation of pathology and important opportunities for discussions regarding management of colorectal conditions. The roles of biopsy, polypectomy, follow-ups, and