Symposium on Diseases of the Liver
Pathogenesis and Therapy of Cholesterol Gallstones With Emphasis on the Metabolic Effects of Chenodeoxycholic Acid
Dean R. Conley, M.D.,* and Leonard 1. Goldstein, MD. **
It is estimated that at least 16 million Americans have gallstones with an additional 800,000 new cases being diagnosed annually. More than 350,000 cholecystectomies are performed yearly, and hospital and related expenses total almost one billion dollars.19 The complications of the disease directly or indirectly result in 6,000 fatalities yearly. The incidence of cholelithiasis increases with age: 30 per cent of the population over the age of 65 have cholelithiasis. In all age groups there is a female-to-male predominance with a ratio of 3:1. These facts tend to underscore the magnitude of the problem of gallstone disease in the United States. The definitive treatment of cholelithiasis, until recently, has been cholecystectomy. However, research advances in the pathogenesis of cholesterol gallstones have placed us on the threshold of a new era, promising medical treatment and prophylaxis of cholelithiasis. The initial report by Danzinger and colleagues9 described chenodeoxycholic acid therapy in 7 women who had asyr1J.ptomatic radiolucent gallstones visualized by oral cholecystography. Chenodeoxycholic acid was administered orally at a dosage of 750 to 4500 mg per day for 6 to 24 months. In all subjects the stones either disappeared or significantly decreased in size. In one patient, 3 stones known to be present for at least 6 years disappeared after 6 months of treatment. Bell et al. 4 treated 22 patients with gallstones. In 19 of these patients, the gallbladders visualized with cholecystography. Gallstone size was assessed prior to therapy and 6 months later. After treatment, the stones disappeared in 4 patients and were reduced in size in 4 others. None of the 9 patients with radiopaque stones responded to treatment. "Postgraduate Fellow in Gastroenterology. Cedars-Sinai Medical Center, Los Angeles, California ** Assistant Director, Gastroenterology, Cedars· Sinai Medical Center, Los Angeles, California
Medical Clinics of North America- VoL 59, No. 4, July 1975
1025
1026
DEAN
R.
CONLEY AND LEONARD
I.
GOLDSTEIN
Thistle and Hofmann 36 reported the results of a randomized, controlled trial using cholic acid, chenodeoxycholic acid, and a placebo in 54 patients with asymptomatic radiolucent stones and visualized gallbladders. Cholecystograms were performed initially and after 6 months of treatment. The gallstones in 12 of the 19 patients receiving chenodeoxycholic acid decreased in size. Cholic acid and placebo were not effective. The absence of response in the placebo group confirms the rarity of spontaneous dissolution of gallstones. More recently, Coyne et al. 7 have published their data on a controlled trial of chenodeoxycholic acid in 20 patients with asymptomatic radiolucent stones. After 1 year of treatment with chenodeoxycholic acid alone, or in combination with phenobarbital, a decrease in gallstone size of more than 50 per cent occurred in 9 of the 20 patients. In 2 of those patients there was complete disappearance of the gallstones (Fig. 1).
At a workshop on chenodeoxycholic acid treatment of gallstones held in Freiburg, Germany in October 1973,17 27 investigators reported their cumulative experience. After six months of chenodeoxycholic acid treatment in doses ranging from 500 to 2000 mg per day, 25 per cent of 76 patients had decreased stone size, and in 6.6 per cent the stones had dissolved. In 50 patients in whom chenodeoxycholic acid therapy was continued for 1 year, 28 per cent had decreased stone size and in 16 per cent, complete gallstone dissolution had occurred. Gallstone dissolution may require 6 to 24 months of therapy. The recurrence rate after discontinuance of treatment and the need for chronic maintenance have not yet been defined. The results of the clinical trials to date indicate that chenodeoxycholic acid is an effective drug in the treatment of cholesterol gallstone disease. Further assessments of the safety and efficacy of chenodeoxycholic acid are currently being evaluated by the Multicenter National Cooperative Gallstone Dissolution Study.
Figure 1. A, Multiple radiolucent gallstones prior to therapy. B, Dissolution of gallstones after one year of treatment with chenodeoxycholic acid (750 mg per day).
P ATHOGENESIS
AND THERAPY OF CHOLESTEROL GALLSTONES
1027
Our aim in this report is to briefly review the pathogenesis of cholesterol stone formation with major emphasis on the metabolic and clinical side effects of chenodeoxycholic acid administration.
PATHOGENESIS OF CHOLESTEROL GALLSTONE FORMATION Ninety per cent of the gallstones analyzed in the United States are composed predominantly of cholesterol in a pure form or cholesterol mixed with bile acid, bile pigments, calcium salts, and protein. The remaining 10 per cent of gallstones are pigment stones. Cholesterol gallstones formation can be conceptually depicted as occurring in three states: saturation, crystallization, and growth. SATURATION. This critical stage results from an alteration of biliary lipids, resulting in a bile saturated with cholesterol. Normally, cholesterol is solubilized in bile by molecular association with bile acids and phospholipids. The predominant phospholipid in bile is lecithin. The water-soluble bile acids incorporate cholesterol-lecithin liquid crystals into molecular aggregates called mixed micelles. Bile that is saturated or contains an excess of cholesterol relative to other biliary lipids is prerequisite for gallstone formation and has been termed "litho genic. " Fifty to 60 per cent of gallstone patients have saturated bile,33 and saturated bile is more prevalent in groups at risk for subsequent development of gallstones. Such groups include young American Indian women,37 (70 per cent of whom have gallstones by age 30), and siblings of young patients with gallstones.s Vlahcevic et al. 39 found no significant difference between the biliary lipid composition of gallbladder bile and hepatic bile in patients with cholesterol gallstones, demonstrating that the liver is the source of saturated bile. Small and R apo35 reached the same conclusion by similar investigations in the American Indian of the Southwest. CRYSTALLIZATION. In this state, cholesterol crystallizes and precipitates from superstaturated bile resulting in two physical states: a solid phase, consisting of cholesterol crystals, and a liquid phase, consisting of bile saturated with cholesterol. Factors responsible for nidus formation include presence of bile pigments, mucoproteins, bacteria, and refluxed intestinal contents. GROWTH. This final stage in the genesis of cholelithiasis begins with the formation of microscopic stones. The rate of growth of these microscopic stones is dependent upon the rate of precipitation on one hand, and the rate of spontaneous dissolution or passage into the intestinal tract on the other. Gallstones usually grow in the gallbladder where stasis, frequency, and efficiency of gallbladder emptying play an additional role. Current5, 25 studies support the concept of a dual mechanism responsible for the hepatic production of litho genic bile - the resultant effect of decreased synthesis and secretion of bile acids and the increased synthesis and secretion of cholesterol. 12
1028
DEAN
R.
CONLEY AND LEONARD
1.
GOLDSTEIN
BILE ACID METABOLISM IN GALLSTONE DISEASE Cholesterol is the obligatory precursor of the bile acids, and the formation of bile acids is a major route of cholesterol elimination from the body. Hydroxylation of cholesterol at the 7 a position of the sterol nucleus by hepatic microsomal cholesterol 7 a hydroxylase is the rate limiting step in bile synthesis. 25 The rate of bile acid synthesis is controlled by a negative feedback mechanism, whereby the rate of synthesis varies inversely with the rate of return of bile acids to the liver. Myant and co-workers! demonstrated enhancement of 7 a hydroxylase activity with interruption of the enterohepatic circulation by dietary diversion. Conversely, feeding rats,28 hamsters,:Jo and human beings,5 primary bile acids (particularly chenodeoxycholic acid) has resulted in inhibition of 7 a hydroxylase activity. Total bile acid pool size in normal people ranges from 2 to 4 gm. The reabsorption of the primary bile salts by the terminal ileum preserves the bile acid pool for repeated utilization. Without an intact enterohepatic circulation, the liver would be unable to augment synthesis enough to maintain the critical micellar concentration in the small intestine that is required for adequate fat solubilization. Patients with cholesterol gallstones have significantly smaller total, cholic, and chenodeoxycholic acid pools that matched controls without stonesYS Pool sizes in patients with cholesterol gallstones have ranged from 397 mg to 2080 mg with a mean pool size of 1235 mg.!O Two mechanisms have been postulated for the decrease bile acid pool size in gallstone patients. One mechanism proposed is that a decrease in the bile acid pool is secondary to an inability to augment hepatic synthesis in the presence of decreased efficiency in bile acid reabsorption. 3s However, there is no published evidence to support that hypothesis. A second mechanism, elucidated by recent experimental data,5.25 supports the concept that defective bile acid synthesis is due to an hepatic enzyme defect. Nicolau et al.2 5 measured hepatic cholesterol 7 a hydroxylase in 4 control subjects and 8 subjects with cholesterol gallstones. Cholesterol 7 a hydroxylase activity averaged 19 ± 2p moles per mg of protein per minute in the controls and 9 ± 2p moles per mg protein per minute in the subjects with gallstones. These results were statistically significant. Bonorris and colleagues5 confirmed Nicolau's findings of a significant reduction in activity of this enzyme when compared to controls. These two concepts are certainly not mutually exclusive and both mechanisms may play a role in the pathogenesis of cholesterol saturated bile. Effects of Chenodeoxycholic Acid on Bile Acid Metabolism Danzinger10 studied the effect of chenodeoxycholic administration on bile acid kinetics in women with cholelithiasis using doses ranging from 750 to 4500 mg per day. They found that the total bile acid pool size increased two-fold with therapy, which reflected an increase in the chenodeoxycholic acid pool and decreases in the cholic and deoxycholic acid pools. Cholic acid synthesis rates in the gallstone patients did not differ from controls in the pretreatment period. However, the cholic acid
P ATHOGENESIS
AND THERAPY OF CHOLESTEROL GALLSTONES
1029
synthesis rate became significantly depressed during chenodeoxycholic acid therapy, demonstrating a negative feedback effect. Analysis of the molar composition of the biliary bile acids reflected the change in composition of the bile acid pool with chenodeoxycholic acid therapy. During treatment, chenodeoxycholic acid comprised approximately 95 per cent of the biliary bile acid. The remaining 5 per cent was composed of cholic acid, deoxycholic acid, and lithocholic acid. The appearance of this latter secondary bile acid, not found during pretreatment biliary drainage, is due to the 7 a dehydroxylation of chenodeoxycholic acid by bacteria in the colon with subsequent absorption of lithocholic acid into the enterohepatic circulation. Approximately 60 per cent of the ingested chenodeoxycholic acid was absorbed and the efficiency of absorption decreased with augmentation of the administered dose. The chenodeoxycholic acid pool appeared to expand in direct proportion to the amount of chenodeoxycholic acid absorbed. In conjunction with a decrease in the synthesis rate of cholic acid was an increased fractional turnover rate of cholic acid, reflecting diminished intestinal conservation. The authors theorized that the conjugation of chenodeoxycholic acid with glycine acts as a competitive inhibitor of cholylglycine transport in the terminal ileum. The resultant effect is a decreased cholic acid pool as a result of the combination of a decreased efficiency of absorption owing to competitive inhibition and decreased synthesis. The negative feedback effect of chenodeoxycholic acid on endogenous bile acid synthesis is further supported by the demonstration of a decline of 47 per cent in the activity of cholesterol 7 a hydroxylase, the rate-limiting enzyme for bile acid synthesis." The simple augmentation of the bile acid pool in patients with cholesterol gallstones is, of itself, not sufficient to reduce cholesterol saturation of the bile. For example, the administration of cholic acid, while increasing the bile acid pool, does not decrease cholesterol saturation, nor is it effective in dissolving gallstones,31 Chenodeoxycholic acid therapy, however, exerts profound effects on lipid metabolism. Thus, the expansion of the bile acid pool combined with alterations in cholesterol metabolism are responsible for the efficacy of chenodeoxycholic acid.
CHOLESTEROL METABOLISM IN GALLSTONE DISEASE Experimental data32 indicates that under basal conditions, bile acids may exert regulatory effects on cholesterol absorption, synthesis, degradation, and excretion. The solubilization of dietary cholesterol for presentation to the intestinal microvilli requires the presence of the amphipathic bile acids. These, in combination with cholesterol, form a mixed micelle allowing hydrolysis of the cholesterol esters on the oil-water interface. Diverting bile acids from the intestinal lumen results in virtually complete cessation of cholesterol absorption. l l The synthesis of hepatic cholesterol from acetyl-CoA is governed by the negative feedback effect of dietary cholesterol modulated by bile acids. The major site of this feedback is in the conversion of BetaHydroxy-Beta-Methyl Glutaryl CoA (HMG-CoA) to mevalonate by the
1030
DEAN
R.
CONLEY AND LEONARD
I.
GOLDSTEIN
enzyme HMG-CoA reductase. 25 Hepatic cholesterol synthesis is markedly enhanced by interference in the enterohepatic circulation of bile acids by external diversion of bile flow,!! or by feeding a bile acid-binding resin, cholestyramine. '8 Ileal bypass operations produce a similar effect on cholesterol synthesis. 24 In addition to a reduction in bile acid synthesis and pool size, cholesterol secretion in bile is increased in Indian and Caucasian women with gallstone disease. g , 37 Increased hepatic synthesis of cholesterol seems to be responsible. Nicolau and colleagues 25 have found, and we have confirmed,5 enhanced HMG CoA reductase activity (the rate limiting enzyme for cholesterol synthesis) in patients with gallstones. Whether these are congenital or acquired metabolic aberrations is not known, But the clustering of gallstone disease in families and among certain ethnic groups suggests that it may be inherited. B,37 In certain species, feeding pharmacologic quantities of bile acids results in accelerated cholesterol absorption. Smalp4 voiced concern about the possible deleterious effects of chenodeoxycholic acid therapy on the lipid metabolism of gallstone patients. He conjectured that chenodeoxycholic acid administration might expand the cholesterol pool, thereby promoting atherosclerosis. Expansion of the cholesterol pool might occur either by facilitation of absorption of cholesterol or by suppression of bile acid synthesis (the major route of cholesterol elimination from the body.) Effects of Chenodeoxycholic Acid on Cholesterol Metabolism Chenodeoxycholic acid administration decreases the secretion of cholesterol in normal subjects. Northfield et alP have showed that chenodeoxycholic acid caused a relative or absolute decrease in the secretion of biliary cholesterol in gallstone patients, with consistent reduction of the cholesterol saturation of bile. This effect may be due to the influence of chenodeoxycholic acid on hepatic enzymatic synthesis of cholesterol. The feeding of chenodeoxycholic acid to hamsters results in a decrease in hepatic HMG-CoA reductase activity by 61 per cent, reflecting a decrease in cholesterol synthesis, while increasing the hepatic bile acid concentration 148 per cent. Moreover, hepatic cholesterol did not increase. 15 Thus, cholesterol synthesis would not seem to be enhanced by such therapy. Chenodeoxycholic acid administration in man produces a similar decrease in hepatic HMG-CoA reductase. 5 Hofmann et al. 16 studied the effects of bile acid feeding on cholesterol metabolism in gallstone patients. They reported that the cholesterol pool size was not altered by chenodeoxycholic acid treatment; nor was there any significant change in serum cholesterol levels after 1 year of treatment. These results confirmed similar observations obtained in the Rhesus monkey after 6 months of treatment,40 and adds little support to the theory that chenodeoxycholic acid therapy is likely to accelerate atherosclerosis. On the contrary, it now appears substantiated2,22 that chenodeoxycholic acid therapy reduces circulating levels of triglycerides. In fact, this has been utilized by Miller and N esteP2 in 11 patients with hypertriglyceridemia associated with primary hyperlipoproteinemias. In 9 of the 11 patients receiving 1 gm of chenodeoxycholic acid per day for 4 weeks, a significant reduction in plasma triglyceride occurred, ranging
P ATHOGENESIS AND THERAPY OF CHOLESTEROL GALLSTONES
1031
from 79 to 986 mg per 100 m!. The magnitude of the reduction correlated with the pre-existing triglyceride concentrations. The mechanism postulated for this occurrence is the inhibition of triglyceride synthesis by the liver. MECHANISMS OF BILE ACID TOXICITY Bile acids are capable of inducing cellular injury through at least two possible mechanisms: (1) the effects of bile acids on cells may be related to their amphipathic structure and resultant ability to interact physiochemically with the cellular lipid membrane. Experimentally, bile acids are capable of lysing the red cell membrane resulting in hemolysis. In this system, chenodeoxycholic acid is less hemolytic than lithocholic and deoxycholic aCids;27 (2) chenodeoxycholic and deoxycholic acids, both dihydroxy bile salts, are able to uncouple oxidative phosphorylation in mitochondria. This toxic mitochondrial injury appears to be inhibited by complete hydroxylation; thus, the trihydroxy bile acid, cholic acid, does not seem to produce this toxic effect. In this regard, Fisher and Phillips4 have noted mitochondrial structural abnormalities in livers perfused with chenodeoxycholic acid. Effects of Chenodeoxycholic Acid on Hepatic Function and Morphology Chenodeoxycholic acid and its metabolite, lithocholic acid, are capable of inducing hepatic injury in a variety of animal species. 26 Although there is some histologic variability among species, bile duct proliferation and fibrosis are common findings. During chenodeoxycholic acid administration in man and in animals, a large quantity, averaging 30 to 40 per cent, is not absorbed by the small intestine and enters the colon. In the colon, colonic bacterial dehydroxylation of the chenodeoxycholic acid molecule occurs, resulting in the formation of lithocholic acid. Lithocholic acid, a monohydroxy bile acid, is water insoluble and thus poorly absorbed. However, some absorption of the lithocholate occurs, permitting its measurement in bile. Biliary lithocholate levels in animals receiving chenodeoxycholic acid have increased from 8 to 14 per cent over the pretreatment value. 15 Similarly, an increase of 2 to 5 per cent is noted in the bile of human beings receiving comparable doses. Lithocholic acid is a potent pyrogen in man and in animals and uniformly produces hepatotoxicity in all species studiedP Therefore, hepatic concentration of lithocholate may result in toxic damage. Normally, lithocholic acid is sulfated by the liver.4 Sulfation facilitates renal excretion and decreases intestinal absorption, serving to protect the liver from its toxic effects. Of particular interest have been the studies of bile acid feeding in primates. Rhesus monkeys, whose bile salt metabolism is similar to humans, were fed chenodeoxycholic acid, 20 mg per kg per day for 15 weeks. 40 SGPT increased 2 to 4-fold in the three animals studied. Liver biopsies showed mild to moderate ductular proliferation with mild inflammation in the portal triads in one animal. The other two animals
1032
DEAN
R.
CONLEY AND LEONARD
I.
GOLDSTEIN
had normal biopsies. A larger study, although uncontrolled, confirmed these findings. '7 These findings in primates contrast dramatically with the development of severe hepatic injury in rabbits, rats, and other lower animals when fed chenodeoxycholic acid. There thus appears to be a marked species variability to the development of hepatotoxicity with chenodeoxycholate administration. It is difficult to relate the results observed in lower species to the potential for toxicity in man. Transient elevations of serum transaminases have been reported in up to 25 per cent of patients during chenodeoxycholic acid treatment. 36 The transaminase elevations return to normal with continuation of therapy. Liver biopsies:; have not shown definite morphologic abnormalities when compared with control patients with cholelithiasis. Coyne and colleagues7 performed liver biopsies on 8 patients with normal liver function tests, receiving chenodeoxycholic acid alone or in combination with phenobarbital. These were compared to liver biopsies obtained in 8 patients receiving placebo or phenobarbital alone. Eleven of the biopsies demonstrated normal hepatic morphology. Four of the remaining five biopsies revealed mild fatty infiltration, only one of the biopsies was from a patient receiving chenodeoxycholic acid. The fifth abnormal biopsy was compatible with alcoholic cirrhosis. Furthermore, the occasional abnormalities found in patients with gallstones during therapy may be due to the presence of cholelithiasis alone, and not related to treatment, since Reichman29 has found some degree of histologic damage in 50 per cent of untreated patients with gallstones. Dowling suggested that the transaminase elevations that are found with chenodeoxycholic acid treatment are dose related. No abnormal liver functions were detected in patients receiving 250 mg of chenodeoxycholic acid per day; but 7 of 27 patlents receiving 750 to 1000 mg per day had an elevation of SGOT on at least one occasion. But, regardless of dose, liver structure was normal in those patients biopsied. Since hepatotoxicity may be dose related in animals40 and man,23 and since the salutory effects on bile lipid composition persist for 20 days after cessation of therapy,'S intermittent or low dose chenodeoxycholic acid treatment for gallstones is being considered. At the present time, the clinical significance and pathophysiologic meaning of the transient elevation in transaminase by chenodeoxycholic acid are unknown. Elevated transaminase activity in the serum may result from a redistribution of transaminase from organelles, chiefly mitochondrial, to the cytosol without necessarily implicating cell destruction. This theory might explain the elevated serum transaminase without evidence of hepatocellular damage. 2o Careful monitoring of other liver function tests by the determination of alkaline phosphatase, BSP retention, serum bilirubin, and albumin has failed to reveal any evidence of hepatic disfunction during treatment.
Effect of Chenodeoxycholic Acid On the Colon Diarrhea induced during chenodeoxycholic acid therapy appears to be dose related. 23 Doses below 750 mg per day rarely produce symptoms.
P ATHOGENESIS
AND THERAPY OF CHOLESTEROL GALLSTONES
1033
In doses over 1000 mg per day, however, mild diarrhea can occur in as many as 40 per cent of patients. Generally, reduction in dose or temporary cessation of therapy will result in amelioration of the symptoms. This diarrhea is presumed to be due to the saturation of the ileal bile salt receptor sites and resultant entry into the colon of chenodeoxycholic acid. The presence of dihydroxy bile acids in the colon inhibits normal colonic absorption of water and electrolytes. Furthermore, net secretion of a bicarbonate-rich fluid results. 21 The interaction of deoxycholic acid, a dihydroxy bile salt, with membrane-bound adenylate cyclase, has been documented by us in vitro 6 and in vivo (unpublished data) with production of colonic secretion. This suggests that the secretion of sodium and water is mediated by cyclic AMP.
CONCLUSION Chenodeoxycholic acid has proven to be an effective drug for the treatment of cholesterol gallstones. Its efficacy is dependent upon its effect on cholesterol and bile acid metabolism. Although it and its metabolite, lithocholic acid, have been found to be hepatotoxic in a variety of species, its effects on primates and human beings is less clear. Evidence that has accumulated to this date indicates that in the dosage employed for gallstone dissolution, chenodeoxycholic acid does not appear to be hepatotoxic in human beings. The development of chenodeoxycholic acid as an effective therapeutic agent for gallstones underscores the tremendous importance of applied research in basic pathophysiology. Cedars-Sinai Medical Center 8720 Beverly Boulevard Los Angeles, California 90048
Pathogenesis and Therapy of Cholesterol Gallstones With Emphasis on the Metabolic Effects of Chenodeoxycholic Acid
Dean R. Conley, M.D.,* and Leonard 1. Goldstein, MD. **
It is estimated that at least 16 million Americans have gallstones with an additional 800,000 new cases being diagnosed annually. More than 350,000 cholecystectomies are performed yearly, and hospital and related expenses total almost one billion dollars.19 The complications of the disease directly or indirectly result in 6,000 fatalities yearly. The incidence of cholelithiasis increases with age: 30 per cent of the population over the age of 65 have cholelithiasis. In all age groups there is a female-to-male predominance with a ratio of 3:1. These facts tend to underscore the magnitude of the problem of gallstone disease in the United States. The definitive treatment of cholelithiasis, until recently, has been cholecystectomy. However, research advances in the pathogenesis of cholesterol gallstones have placed us on the threshold of a new era, promising medical treatment and prophylaxis of cholelithiasis. The initial report by Danzinger and colleagues9 described chenodeoxycholic acid therapy in 7 women who had asyr1J.ptomatic radiolucent gallstones visualized by oral cholecystography. Chenodeoxycholic acid was administered orally at a dosage of 750 to 4500 mg per day for 6 to 24 months. In all subjects the stones either disappeared or significantly decreased in size. In one patient, 3 stones known to be present for at least 6 years disappeared after 6 months of treatment. Bell et al. 4 treated 22 patients with gallstones. In 19 of these patients, the gallbladders visualized with cholecystography. Gallstone size was assessed prior to therapy and 6 months later. After treatment, the stones disappeared in 4 patients and were reduced in size in 4 others. None of the 9 patients with radiopaque stones responded to treatment. "Postgraduate Fellow in Gastroenterology. Cedars-Sinai Medical Center, Los Angeles, California ** Assistant Director, Gastroenterology, Cedars· Sinai Medical Center, Los Angeles, California
Medical Clinics of North America- VoL 59, No. 4, July 1975
1025
1026
DEAN
R.
CONLEY AND LEONARD
I.
GOLDSTEIN
Thistle and Hofmann 36 reported the results of a randomized, controlled trial using cholic acid, chenodeoxycholic acid, and a placebo in 54 patients with asymptomatic radiolucent stones and visualized gallbladders. Cholecystograms were performed initially and after 6 months of treatment. The gallstones in 12 of the 19 patients receiving chenodeoxycholic acid decreased in size. Cholic acid and placebo were not effective. The absence of response in the placebo group confirms the rarity of spontaneous dissolution of gallstones. More recently, Coyne et al. 7 have published their data on a controlled trial of chenodeoxycholic acid in 20 patients with asymptomatic radiolucent stones. After 1 year of treatment with chenodeoxycholic acid alone, or in combination with phenobarbital, a decrease in gallstone size of more than 50 per cent occurred in 9 of the 20 patients. In 2 of those patients there was complete disappearance of the gallstones (Fig. 1).
At a workshop on chenodeoxycholic acid treatment of gallstones held in Freiburg, Germany in October 1973,17 27 investigators reported their cumulative experience. After six months of chenodeoxycholic acid treatment in doses ranging from 500 to 2000 mg per day, 25 per cent of 76 patients had decreased stone size, and in 6.6 per cent the stones had dissolved. In 50 patients in whom chenodeoxycholic acid therapy was continued for 1 year, 28 per cent had decreased stone size and in 16 per cent, complete gallstone dissolution had occurred. Gallstone dissolution may require 6 to 24 months of therapy. The recurrence rate after discontinuance of treatment and the need for chronic maintenance have not yet been defined. The results of the clinical trials to date indicate that chenodeoxycholic acid is an effective drug in the treatment of cholesterol gallstone disease. Further assessments of the safety and efficacy of chenodeoxycholic acid are currently being evaluated by the Multicenter National Cooperative Gallstone Dissolution Study.
Figure 1. A, Multiple radiolucent gallstones prior to therapy. B, Dissolution of gallstones after one year of treatment with chenodeoxycholic acid (750 mg per day).
P ATHOGENESIS
AND THERAPY OF CHOLESTEROL GALLSTONES
1027
Our aim in this report is to briefly review the pathogenesis of cholesterol stone formation with major emphasis on the metabolic and clinical side effects of chenodeoxycholic acid administration.
PATHOGENESIS OF CHOLESTEROL GALLSTONE FORMATION Ninety per cent of the gallstones analyzed in the United States are composed predominantly of cholesterol in a pure form or cholesterol mixed with bile acid, bile pigments, calcium salts, and protein. The remaining 10 per cent of gallstones are pigment stones. Cholesterol gallstones formation can be conceptually depicted as occurring in three states: saturation, crystallization, and growth. SATURATION. This critical stage results from an alteration of biliary lipids, resulting in a bile saturated with cholesterol. Normally, cholesterol is solubilized in bile by molecular association with bile acids and phospholipids. The predominant phospholipid in bile is lecithin. The water-soluble bile acids incorporate cholesterol-lecithin liquid crystals into molecular aggregates called mixed micelles. Bile that is saturated or contains an excess of cholesterol relative to other biliary lipids is prerequisite for gallstone formation and has been termed "litho genic. " Fifty to 60 per cent of gallstone patients have saturated bile,33 and saturated bile is more prevalent in groups at risk for subsequent development of gallstones. Such groups include young American Indian women,37 (70 per cent of whom have gallstones by age 30), and siblings of young patients with gallstones.s Vlahcevic et al. 39 found no significant difference between the biliary lipid composition of gallbladder bile and hepatic bile in patients with cholesterol gallstones, demonstrating that the liver is the source of saturated bile. Small and R apo35 reached the same conclusion by similar investigations in the American Indian of the Southwest. CRYSTALLIZATION. In this state, cholesterol crystallizes and precipitates from superstaturated bile resulting in two physical states: a solid phase, consisting of cholesterol crystals, and a liquid phase, consisting of bile saturated with cholesterol. Factors responsible for nidus formation include presence of bile pigments, mucoproteins, bacteria, and refluxed intestinal contents. GROWTH. This final stage in the genesis of cholelithiasis begins with the formation of microscopic stones. The rate of growth of these microscopic stones is dependent upon the rate of precipitation on one hand, and the rate of spontaneous dissolution or passage into the intestinal tract on the other. Gallstones usually grow in the gallbladder where stasis, frequency, and efficiency of gallbladder emptying play an additional role. Current5, 25 studies support the concept of a dual mechanism responsible for the hepatic production of litho genic bile - the resultant effect of decreased synthesis and secretion of bile acids and the increased synthesis and secretion of cholesterol. 12
1028
DEAN
R.
CONLEY AND LEONARD
1.
GOLDSTEIN
BILE ACID METABOLISM IN GALLSTONE DISEASE Cholesterol is the obligatory precursor of the bile acids, and the formation of bile acids is a major route of cholesterol elimination from the body. Hydroxylation of cholesterol at the 7 a position of the sterol nucleus by hepatic microsomal cholesterol 7 a hydroxylase is the rate limiting step in bile synthesis. 25 The rate of bile acid synthesis is controlled by a negative feedback mechanism, whereby the rate of synthesis varies inversely with the rate of return of bile acids to the liver. Myant and co-workers! demonstrated enhancement of 7 a hydroxylase activity with interruption of the enterohepatic circulation by dietary diversion. Conversely, feeding rats,28 hamsters,:Jo and human beings,5 primary bile acids (particularly chenodeoxycholic acid) has resulted in inhibition of 7 a hydroxylase activity. Total bile acid pool size in normal people ranges from 2 to 4 gm. The reabsorption of the primary bile salts by the terminal ileum preserves the bile acid pool for repeated utilization. Without an intact enterohepatic circulation, the liver would be unable to augment synthesis enough to maintain the critical micellar concentration in the small intestine that is required for adequate fat solubilization. Patients with cholesterol gallstones have significantly smaller total, cholic, and chenodeoxycholic acid pools that matched controls without stonesYS Pool sizes in patients with cholesterol gallstones have ranged from 397 mg to 2080 mg with a mean pool size of 1235 mg.!O Two mechanisms have been postulated for the decrease bile acid pool size in gallstone patients. One mechanism proposed is that a decrease in the bile acid pool is secondary to an inability to augment hepatic synthesis in the presence of decreased efficiency in bile acid reabsorption. 3s However, there is no published evidence to support that hypothesis. A second mechanism, elucidated by recent experimental data,5.25 supports the concept that defective bile acid synthesis is due to an hepatic enzyme defect. Nicolau et al.2 5 measured hepatic cholesterol 7 a hydroxylase in 4 control subjects and 8 subjects with cholesterol gallstones. Cholesterol 7 a hydroxylase activity averaged 19 ± 2p moles per mg of protein per minute in the controls and 9 ± 2p moles per mg protein per minute in the subjects with gallstones. These results were statistically significant. Bonorris and colleagues5 confirmed Nicolau's findings of a significant reduction in activity of this enzyme when compared to controls. These two concepts are certainly not mutually exclusive and both mechanisms may play a role in the pathogenesis of cholesterol saturated bile. Effects of Chenodeoxycholic Acid on Bile Acid Metabolism Danzinger10 studied the effect of chenodeoxycholic administration on bile acid kinetics in women with cholelithiasis using doses ranging from 750 to 4500 mg per day. They found that the total bile acid pool size increased two-fold with therapy, which reflected an increase in the chenodeoxycholic acid pool and decreases in the cholic and deoxycholic acid pools. Cholic acid synthesis rates in the gallstone patients did not differ from controls in the pretreatment period. However, the cholic acid
P ATHOGENESIS
AND THERAPY OF CHOLESTEROL GALLSTONES
1029
synthesis rate became significantly depressed during chenodeoxycholic acid therapy, demonstrating a negative feedback effect. Analysis of the molar composition of the biliary bile acids reflected the change in composition of the bile acid pool with chenodeoxycholic acid therapy. During treatment, chenodeoxycholic acid comprised approximately 95 per cent of the biliary bile acid. The remaining 5 per cent was composed of cholic acid, deoxycholic acid, and lithocholic acid. The appearance of this latter secondary bile acid, not found during pretreatment biliary drainage, is due to the 7 a dehydroxylation of chenodeoxycholic acid by bacteria in the colon with subsequent absorption of lithocholic acid into the enterohepatic circulation. Approximately 60 per cent of the ingested chenodeoxycholic acid was absorbed and the efficiency of absorption decreased with augmentation of the administered dose. The chenodeoxycholic acid pool appeared to expand in direct proportion to the amount of chenodeoxycholic acid absorbed. In conjunction with a decrease in the synthesis rate of cholic acid was an increased fractional turnover rate of cholic acid, reflecting diminished intestinal conservation. The authors theorized that the conjugation of chenodeoxycholic acid with glycine acts as a competitive inhibitor of cholylglycine transport in the terminal ileum. The resultant effect is a decreased cholic acid pool as a result of the combination of a decreased efficiency of absorption owing to competitive inhibition and decreased synthesis. The negative feedback effect of chenodeoxycholic acid on endogenous bile acid synthesis is further supported by the demonstration of a decline of 47 per cent in the activity of cholesterol 7 a hydroxylase, the rate-limiting enzyme for bile acid synthesis." The simple augmentation of the bile acid pool in patients with cholesterol gallstones is, of itself, not sufficient to reduce cholesterol saturation of the bile. For example, the administration of cholic acid, while increasing the bile acid pool, does not decrease cholesterol saturation, nor is it effective in dissolving gallstones,31 Chenodeoxycholic acid therapy, however, exerts profound effects on lipid metabolism. Thus, the expansion of the bile acid pool combined with alterations in cholesterol metabolism are responsible for the efficacy of chenodeoxycholic acid.
CHOLESTEROL METABOLISM IN GALLSTONE DISEASE Experimental data32 indicates that under basal conditions, bile acids may exert regulatory effects on cholesterol absorption, synthesis, degradation, and excretion. The solubilization of dietary cholesterol for presentation to the intestinal microvilli requires the presence of the amphipathic bile acids. These, in combination with cholesterol, form a mixed micelle allowing hydrolysis of the cholesterol esters on the oil-water interface. Diverting bile acids from the intestinal lumen results in virtually complete cessation of cholesterol absorption. l l The synthesis of hepatic cholesterol from acetyl-CoA is governed by the negative feedback effect of dietary cholesterol modulated by bile acids. The major site of this feedback is in the conversion of BetaHydroxy-Beta-Methyl Glutaryl CoA (HMG-CoA) to mevalonate by the
1030
DEAN
R.
CONLEY AND LEONARD
I.
GOLDSTEIN
enzyme HMG-CoA reductase. 25 Hepatic cholesterol synthesis is markedly enhanced by interference in the enterohepatic circulation of bile acids by external diversion of bile flow,!! or by feeding a bile acid-binding resin, cholestyramine. '8 Ileal bypass operations produce a similar effect on cholesterol synthesis. 24 In addition to a reduction in bile acid synthesis and pool size, cholesterol secretion in bile is increased in Indian and Caucasian women with gallstone disease. g , 37 Increased hepatic synthesis of cholesterol seems to be responsible. Nicolau and colleagues 25 have found, and we have confirmed,5 enhanced HMG CoA reductase activity (the rate limiting enzyme for cholesterol synthesis) in patients with gallstones. Whether these are congenital or acquired metabolic aberrations is not known, But the clustering of gallstone disease in families and among certain ethnic groups suggests that it may be inherited. B,37 In certain species, feeding pharmacologic quantities of bile acids results in accelerated cholesterol absorption. Smalp4 voiced concern about the possible deleterious effects of chenodeoxycholic acid therapy on the lipid metabolism of gallstone patients. He conjectured that chenodeoxycholic acid administration might expand the cholesterol pool, thereby promoting atherosclerosis. Expansion of the cholesterol pool might occur either by facilitation of absorption of cholesterol or by suppression of bile acid synthesis (the major route of cholesterol elimination from the body.) Effects of Chenodeoxycholic Acid on Cholesterol Metabolism Chenodeoxycholic acid administration decreases the secretion of cholesterol in normal subjects. Northfield et alP have showed that chenodeoxycholic acid caused a relative or absolute decrease in the secretion of biliary cholesterol in gallstone patients, with consistent reduction of the cholesterol saturation of bile. This effect may be due to the influence of chenodeoxycholic acid on hepatic enzymatic synthesis of cholesterol. The feeding of chenodeoxycholic acid to hamsters results in a decrease in hepatic HMG-CoA reductase activity by 61 per cent, reflecting a decrease in cholesterol synthesis, while increasing the hepatic bile acid concentration 148 per cent. Moreover, hepatic cholesterol did not increase. 15 Thus, cholesterol synthesis would not seem to be enhanced by such therapy. Chenodeoxycholic acid administration in man produces a similar decrease in hepatic HMG-CoA reductase. 5 Hofmann et al. 16 studied the effects of bile acid feeding on cholesterol metabolism in gallstone patients. They reported that the cholesterol pool size was not altered by chenodeoxycholic acid treatment; nor was there any significant change in serum cholesterol levels after 1 year of treatment. These results confirmed similar observations obtained in the Rhesus monkey after 6 months of treatment,40 and adds little support to the theory that chenodeoxycholic acid therapy is likely to accelerate atherosclerosis. On the contrary, it now appears substantiated2,22 that chenodeoxycholic acid therapy reduces circulating levels of triglycerides. In fact, this has been utilized by Miller and N esteP2 in 11 patients with hypertriglyceridemia associated with primary hyperlipoproteinemias. In 9 of the 11 patients receiving 1 gm of chenodeoxycholic acid per day for 4 weeks, a significant reduction in plasma triglyceride occurred, ranging
P ATHOGENESIS AND THERAPY OF CHOLESTEROL GALLSTONES
1031
from 79 to 986 mg per 100 m!. The magnitude of the reduction correlated with the pre-existing triglyceride concentrations. The mechanism postulated for this occurrence is the inhibition of triglyceride synthesis by the liver. MECHANISMS OF BILE ACID TOXICITY Bile acids are capable of inducing cellular injury through at least two possible mechanisms: (1) the effects of bile acids on cells may be related to their amphipathic structure and resultant ability to interact physiochemically with the cellular lipid membrane. Experimentally, bile acids are capable of lysing the red cell membrane resulting in hemolysis. In this system, chenodeoxycholic acid is less hemolytic than lithocholic and deoxycholic aCids;27 (2) chenodeoxycholic and deoxycholic acids, both dihydroxy bile salts, are able to uncouple oxidative phosphorylation in mitochondria. This toxic mitochondrial injury appears to be inhibited by complete hydroxylation; thus, the trihydroxy bile acid, cholic acid, does not seem to produce this toxic effect. In this regard, Fisher and Phillips4 have noted mitochondrial structural abnormalities in livers perfused with chenodeoxycholic acid. Effects of Chenodeoxycholic Acid on Hepatic Function and Morphology Chenodeoxycholic acid and its metabolite, lithocholic acid, are capable of inducing hepatic injury in a variety of animal species. 26 Although there is some histologic variability among species, bile duct proliferation and fibrosis are common findings. During chenodeoxycholic acid administration in man and in animals, a large quantity, averaging 30 to 40 per cent, is not absorbed by the small intestine and enters the colon. In the colon, colonic bacterial dehydroxylation of the chenodeoxycholic acid molecule occurs, resulting in the formation of lithocholic acid. Lithocholic acid, a monohydroxy bile acid, is water insoluble and thus poorly absorbed. However, some absorption of the lithocholate occurs, permitting its measurement in bile. Biliary lithocholate levels in animals receiving chenodeoxycholic acid have increased from 8 to 14 per cent over the pretreatment value. 15 Similarly, an increase of 2 to 5 per cent is noted in the bile of human beings receiving comparable doses. Lithocholic acid is a potent pyrogen in man and in animals and uniformly produces hepatotoxicity in all species studiedP Therefore, hepatic concentration of lithocholate may result in toxic damage. Normally, lithocholic acid is sulfated by the liver.4 Sulfation facilitates renal excretion and decreases intestinal absorption, serving to protect the liver from its toxic effects. Of particular interest have been the studies of bile acid feeding in primates. Rhesus monkeys, whose bile salt metabolism is similar to humans, were fed chenodeoxycholic acid, 20 mg per kg per day for 15 weeks. 40 SGPT increased 2 to 4-fold in the three animals studied. Liver biopsies showed mild to moderate ductular proliferation with mild inflammation in the portal triads in one animal. The other two animals
1032
DEAN
R.
CONLEY AND LEONARD
I.
GOLDSTEIN
had normal biopsies. A larger study, although uncontrolled, confirmed these findings. '7 These findings in primates contrast dramatically with the development of severe hepatic injury in rabbits, rats, and other lower animals when fed chenodeoxycholic acid. There thus appears to be a marked species variability to the development of hepatotoxicity with chenodeoxycholate administration. It is difficult to relate the results observed in lower species to the potential for toxicity in man. Transient elevations of serum transaminases have been reported in up to 25 per cent of patients during chenodeoxycholic acid treatment. 36 The transaminase elevations return to normal with continuation of therapy. Liver biopsies:; have not shown definite morphologic abnormalities when compared with control patients with cholelithiasis. Coyne and colleagues7 performed liver biopsies on 8 patients with normal liver function tests, receiving chenodeoxycholic acid alone or in combination with phenobarbital. These were compared to liver biopsies obtained in 8 patients receiving placebo or phenobarbital alone. Eleven of the biopsies demonstrated normal hepatic morphology. Four of the remaining five biopsies revealed mild fatty infiltration, only one of the biopsies was from a patient receiving chenodeoxycholic acid. The fifth abnormal biopsy was compatible with alcoholic cirrhosis. Furthermore, the occasional abnormalities found in patients with gallstones during therapy may be due to the presence of cholelithiasis alone, and not related to treatment, since Reichman29 has found some degree of histologic damage in 50 per cent of untreated patients with gallstones. Dowling suggested that the transaminase elevations that are found with chenodeoxycholic acid treatment are dose related. No abnormal liver functions were detected in patients receiving 250 mg of chenodeoxycholic acid per day; but 7 of 27 patlents receiving 750 to 1000 mg per day had an elevation of SGOT on at least one occasion. But, regardless of dose, liver structure was normal in those patients biopsied. Since hepatotoxicity may be dose related in animals40 and man,23 and since the salutory effects on bile lipid composition persist for 20 days after cessation of therapy,'S intermittent or low dose chenodeoxycholic acid treatment for gallstones is being considered. At the present time, the clinical significance and pathophysiologic meaning of the transient elevation in transaminase by chenodeoxycholic acid are unknown. Elevated transaminase activity in the serum may result from a redistribution of transaminase from organelles, chiefly mitochondrial, to the cytosol without necessarily implicating cell destruction. This theory might explain the elevated serum transaminase without evidence of hepatocellular damage. 2o Careful monitoring of other liver function tests by the determination of alkaline phosphatase, BSP retention, serum bilirubin, and albumin has failed to reveal any evidence of hepatic disfunction during treatment.
Effect of Chenodeoxycholic Acid On the Colon Diarrhea induced during chenodeoxycholic acid therapy appears to be dose related. 23 Doses below 750 mg per day rarely produce symptoms.
P ATHOGENESIS
AND THERAPY OF CHOLESTEROL GALLSTONES
1033
In doses over 1000 mg per day, however, mild diarrhea can occur in as many as 40 per cent of patients. Generally, reduction in dose or temporary cessation of therapy will result in amelioration of the symptoms. This diarrhea is presumed to be due to the saturation of the ileal bile salt receptor sites and resultant entry into the colon of chenodeoxycholic acid. The presence of dihydroxy bile acids in the colon inhibits normal colonic absorption of water and electrolytes. Furthermore, net secretion of a bicarbonate-rich fluid results. 21 The interaction of deoxycholic acid, a dihydroxy bile salt, with membrane-bound adenylate cyclase, has been documented by us in vitro 6 and in vivo (unpublished data) with production of colonic secretion. This suggests that the secretion of sodium and water is mediated by cyclic AMP.
CONCLUSION Chenodeoxycholic acid has proven to be an effective drug for the treatment of cholesterol gallstones. Its efficacy is dependent upon its effect on cholesterol and bile acid metabolism. Although it and its metabolite, lithocholic acid, have been found to be hepatotoxic in a variety of species, its effects on primates and human beings is less clear. Evidence that has accumulated to this date indicates that in the dosage employed for gallstone dissolution, chenodeoxycholic acid does not appear to be hepatotoxic in human beings. The development of chenodeoxycholic acid as an effective therapeutic agent for gallstones underscores the tremendous importance of applied research in basic pathophysiology. Cedars-Sinai Medical Center 8720 Beverly Boulevard Los Angeles, California 90048
