LIVER PHYSIOLOGY AND DISEASE

Transcription

1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.2 Printed in U.S.A. LIVER PHYSIOLOGY AND DISEASE BILE ACID METABOLISM IN CIRRHOSIS III. Biliary lipid secretion in patients with cirrhosis and its relevance to gallstone formation Z. R. VLAHCEVIC, M.D., T. YOSHIDA, M.D., P. JUTTIJUDATA, M.D., C. C. BELL, JR., M.D., AND LEON SWELL, PH.D. Veterans Administration Hospital, and Departments of Medicine (Division of Gastroenterology), Surgery, and Biochemistry, Virginia Commonwealth University, Health Sciences Division, Richmond, Virginia Biliary lipid secretion and bile acid pools were determined in 10 patients without and 13 patients with cirrhosis. Patients with cirrhosis were found to have a greatly diminished total bile acid pool. This alteration in the bile acid pool was associated with a significantly decreased phospholipid secretion, and a very marked decrease in biliary cholesterol secretion. The concentration of the biliary lipids in duodenal bile paralleled the changes in biliary lipid secretion. A triangular phase diagram plot of the biliary lipid composition data indicated that the bile of patients with cirrhosis was entirely normal (no insoluble cholesterol) and had a greater cholesterol holding capacity than the bile of control subjects. These findings indicate that cirrhotic patients are not predisposed to develop cholesterol gallstones. The reported high incidence of gallstones in cirrhotic patients may be related to an abnormality in bilirubin metabolism since a high proportion of the stones in these patients have been reported to be of the pigmented type. Recent reports 1-3 have shown that both Caucasians and American Indians with cholesterol gallstones have a diminished bile acid pool. This decrease in the bile acid pool could be an important factor Received June 12, Accepted September 1, Address requests for reprints to: Dr. Z. R. Vlahcevic, Medical Service, Division of Gastroenterology, Veterans Administration Hospital, Richmond, Virginia This work was supported in part by United States Public Health Service Research Grant 1 ROl-AM from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service. 298 initiating the development of cholesterol gallstones in man. Evidence 4 has also been presented to show that the decrease in the bile acid pool of patients with gallstones leads to a decrease in phospholipid secretion with no significant change in cholesterol secretion. These alterations in biliary lipid secretion could produce a relative decrease in bile salts and phospholipid in relation to cholesterol creating the conditions favorable for the precipitation of biliary cholesterol. The recent observations 5. 6 that patients with regional en teritis, ileal resection, or bypass have a high incidence of cholesterol gallstones, also lend support to the view that a diminished

2 February 1973 LIVER PHYSIOLOGY AND DISEASE 299 bile acid pool may play an important role in the etiology of cholesterol gallstones in man. A greatly diminished bile acid pool has also been observed in patients with cirrhosis. 7 If a reduced bile acid pool is the sole factor leading to the development of cholesterol gallstones, then these patients should also have a lithogenic bile and high incidence of cholesterol gallstones. Recent reports 8, 9 have noted that gallstones are more common in patients with cirrhosis than in the general population. However, in contrast to the general population where the majority of gallstones are of the cholesterol type, cirrhotic patients were found to have principally pigmented or calcium bilirubinate stones. We have also noted that the gallstones obtained from several cirrhotic patients contained virtually no cholesterol and were of the pigmented type. These observations raise the important question as to why a decreased bile acid pool in these patients is not associated with the development of cholesterol gallstones. Patients with cirrhosis may not have a lithogenic bile, either due to an increase in phospholipid or a decrease in cholesterol secretion. The purpose of this study was to obtain definitive information on the interrelationship between the physicochemical state of the bile and biliary lipid secretion in cirrhotic patients. Experimental Procedures Patients. Two groups of male patients ranging in age from 37 to 72 years were studied. The groups consisted of 10 patients without evidence of gallstones or liver disease and a group of 13 cirrhotic patients. The presence or absence of gallstones in control patients was determined by oral cholecystogram. The pertinent clinical data on these patients have been reported earlier. 10, 11 Bile acid pools were determined on each patient after the administration of "C-cholic and "C-chenodeoxycholic acids, Bile-rich duodenal fluid was obtained by intubation with a Dreiling double lumen tube after the administration of 40 Ivy units of cholecystokinin administered intravenously. A small portion (5 ml) of the bilerich duodenal fluid was obtained for analysis and the remainder was returned to the patient via the tube. Duodenal bile samples were obtained on 4 to 5 consecutive days after the administration of the labeled bile acids. The duodenal bile was immersed in ice and extracted within 15 min. Methods. Bile-rich duodenal fluid was brought to room temperature, agitated for 2 min and then extracted with 20 volumes of 2: 1 chloroform-methanol and partitioned with 0.2 volume of water. 12 Bile acid analyses were carried out on the upper methanol-water phase as described earlier. 1, 10 The biliary lipids (phospholipid and cholesterol) were extracted into the chloroform phase. Phospholipid phosphorus was determined by the method of Bartlett'3 and cholesterol as the digitonide by the method of Sperry and Webb." Calculations. The component and total bile acid pools on the patients were estimated from the cholic and chenodeoxycholic acid specific activity decay curves, the amount of administered "C-bile acids, and the bile acid composition. 'O Biliary lipid secretion per bile acid pool was estimated as reported earlier.4 Total lipid secretion per bile acid pool was calculated by dividing the fraction of the bile salts in the total biliary lipid mixture into the total bile acid pool in micromoles. The amounts of phospholipid and cholesterol secreted in micromoles were calculated from the total biliary lipid secreted and the percentage of cholesterol and phospholipid in the total lipid mixture. These data represent the phospholipid and cholesterol secreted with a patient's total bile acid pool. The basis for the estimation of the quantity of phospholipid and cholesterol secreted per total bile acid pool or cycle has previously been presented. 4 A critical factor iri this calculation is the constancy of lipid composition of duodenal bile. In order to validate this procedure more fully for the estimation of biliary lipid secretion, experiments were carried out to ascertain the diurnal variation of the gallbladder bile in a series of patients with and without liver disease. These data are shown in table 1. Bile samples were obtained at 8 AM, 3 PM, and 8 PM during a single day. The 8 AM collection was after an overnight fast. The patients were then allowed to have their meals and bile samples were collected 2 to 3 hr after eating. The ratios of bile salts to phospholipid and bile salts to cholesterol remained constant throughout the day in patients with and without liver disease. No significant differences were noted (paired t-test) in the ratio of the excreted biliary lipids between morning and evening, morning and

3 300 LIVER PHYSIOLOGY AND DISEASE Vol. 64, No.2 TABLE 1. Diurnal variation of duodenal biliary lipids Biliary lipid ratios Group No. of patients Bile salts-phospholipid a Bile salts-cholesterola SAM 3 PM 8 PM SAM 3 PM S PM No cirrhosis ±1.09c ±1.06 ±0.92 ±6.71 ±6.24 ±7.37 Cirrhosis.., ±0.83 ±0.58 ±0.51 ±2.17 ±2.43 ± a Paired t-test comparisons showed no significant differences (P > 0.05) In the biliary lipid ratios as a function of time within each group of patients. Ratios were derived from concentration data. b Patients with no cirrhosis had normal liver function and no evidence of gallstones. c Represents ± standard deviation. afternoon, and afternoon and evening. These data suggest that the duodenal bile obtained after con traction of the gallbladder by cholecystokinin has a relatively constant biliary lipid composition in a given individual throughout the day. The data were processed in an IBM 370 computer. Appropriate programs were written to calculate the data and carry out statistical analyses. Results The biliary lipid composition data were plotted on triangular coordinates as described by Admirand and Small 15 and are shown in figure 1. These data represent the analysis of four to five samples of daily duodenal bile per patient. The average biliary lipid composition for both groups of patients ± standard deviation are represented in the diagram. The biles of both types of patients were within the micellar zone. However, the bile of patients with cirrhosis was more unsaturated with respect to cholesterol than the bile of the group without cirrhosis. The ratio of the cholesterol-solubilizing agents (bile salts + phospholipid) to cholesterol was significantly increased in patients with cirrhosis (17.7 ± 7.8) as compared with patients without cirrhosis (11.9 ± 2.6). These observations indicate that the bile of both types of patients was normal, but the bile of cirrhotic patients had a greater cholesterol-holding capacity. The amount of biliary phospholipids and cholesterol secreted with one bile acid pool is shown in table 2. The total bile acid pool Ptf'~t8tIfSalt CIRRHOSIS FIG. 1. A triangular phase diagram plot of the biliary lipid composition data of the 10 patients without cirrhosis and 13 patients with cirrhosis. Represents the average ± standard deviation. Each value per patient represents the average of four to five separate biliary lipid composition determinations. The line of maximum cholesterol solubility is taken from the model system described by Admirand and Smal!.' was reduced by about 46% in patients with cirrhosis. The secretion of biliary phospholipids was significantly reduced (about 30%) in patients with cirrhosis. There was a much more marked and highly significant redu.ction, however, in the secretion of biliary cholesterol in these patients. The secretion of cholesterol in the cirrhotic group was about one-third that of the group without cirrhosis; the difference is found to be statistically highly significant. The concentration of biliary lipids expressed in terms of inicromoles per milli-

4 FebT1U1ry 1973 LIVER PHYSIOLOGY AND DISEASE 301 liter is shown in table 3. An average biliary lipid concentration on each patient was obtained by averaging four to five bile samples. The individual values for all patients were then averaged to obtain the average for each group. These data are shown for comparative purposes since the concentrating ability of the gallbladder may have a marked affect on the concentration of the biliary lipids in duodenal bile. However, they do show trends which correlate very well with the data on biliary lipid secretion as shown in table 2. Patients with cirrhosis had a marked reduction in the concentration of all three biliary lipids. However, the concentration of cholesterol was more markedly reduced than any other lipid. Patients with cirrhosis had a cholesterol concentration of about one-eighth that of the normal group. Phospholipid concentration in patients with cirrhosis was about one-fourth that of the group without cirrhosis and bile salt concentration was about one-sixth that of the normal. All of these differences were significant at the p < level. TABLE 2. Biliary lipid secretion Micromoles of lipid secreted per bile acid pool Patient no. Phospholipid Cholesterol Bile salts ~ total pool No cirrhosis Average (lo)a 1816 ± 608" 648 ± ± 1115 Cirrhosis Average 1209 ± ± ± 851 P< P < P< a Represents the average of 10 patlents without hver disease and no evidence of gallstones previously reported. to "Represents ± standard deviation. No. of patients" TABLE 3. Biliary lipid concentration Phospholipid Micromoles per 1 ml of duodenal bile Cholesterol No cirrhosis ± 7.34" 4.96 ± 2.92 Cirrhosis ± ± 0.53 P < P < a Each value per patient represents the average of four to five daily determinations. "Represents ± standard deviation. Bile salts ± ± 6.15 P< 0.001

5 302 LIVER PHYSIOLOGY AND DISEASE Vol. 64, No.2 Discussion The findings of the earlier reports 1-3, have presented evidence that the total bile acid pool is markedly reduced in patients with cholesterol gallstones and also in patients with cirrhosis. The bile acid pool size in both groups of patients was similar (average 1.3 g) which is about 46% lower than that found in normal subjects (2.3 g). It was also shown that the decrease in the bile acid pool of male patients with cholesterol gallstones was associated with a significant reduction in biliary phospholipid secretion, but no significant change in cholesterol secretion. 3, 4 The diminished bile acid pool coupled with a decrease in the secretion of biliary phospholipids and no alteration in cholesterol secretion is probably a major factor contributing to the formation of the lithogenic bile found in patients with cholesterol gallstones. The findings of the present report indicate that a decrease in bile acid pool is not necessarily the sole factor responsible for the formation of lithogenic bile, since the bile of patients with cirrhosis was highly unsaturated in respect to cholesterol. In fact, the cholesterol-solubilizing capacity of the bile from these patients was significantly higher than the bile of the normal subjects. The observed differences in biliary lipid composition between cirrhotic and cholesterol gallstone patients, 3. 4 can be best explained by comparing the patterns of bilirary lipid secretion of these patients. Both groups of patients show similar decreases in phospholipid secretion when compared with normal subjects. However, cholesterol secretion is greatly reduced in cirrhotic patients, but remains unchanged in male patients with cholesterol gallstones. Since the cholesterol secretion was reduced to a much greater extent than bile salt and phospholipids, the bile of cirrhotic patients remained well within the micellar zone. The observed changes in the secretion of the biliary lipids calculated from the bile acid pool and biliary lipid composition data are corroborated by similar parallel changes in the concentration of biliary lipids in cirrhotic and gallstone patients (unpublished data). The pronounced decrease in the secretion of biliary cholesterol in patients with cirrhosis may explain why the gallstones encountered in this type of patient contain very little cholesterol. The reported higher incidence of gallstones in cirrhotic patients 8, 9 is probably associated with a conjugation defect in metabolism of bilirubin which results in an increase of free bilirubin in bile. Such conditions would favor the precipitation of calcium bilirubinate which could lead to the formation of pigmented stones. An intimate relationship between bile acid and phospholipid secretion has been demonstrated in perfused rat 16 and dog liver 17 and in patients with T -tubes. 18 The interdependency between phospholipid and bile acid secretion is also present in cirrhotic, normal, and gallstone subjects with an intact enterohepatic circulation.4, 19 The decrease in concentration of bile acids returning to the liver, which is probably present in patients with reduced bile acid pool, is associated with a concomitant drop in phospholipid secretion. A high degree of correlation between bile salt and phospholipid secretion was also noted for patients with and without cirrhosis which suggests that the common factor responsible for the reduction in biliary phospholipid secretion in cirrhotic patients is the diminished secretion of bile acids by the liver. A decrease in the concentration of bile acids returning to the liver could effect the release of lecithin into the bile 17, 18 and also the synthesis of the biliary lecithin by the liver.20 There is no such consistency with respect to the affect of cholelithiasis or liver disease on the secretion of biliary cholesterol. Biliary cholesterol secretion varies with the type of the disorder and sex, and is not as responsive to changes in bile acid pool size as phospholipid secretion. The decrease in the bile acid pool in male Caucasians and Indian patients with cholesterol gallstones is not associated with a change in cholesterol secretion. 1, 3, 4 On the other hand, the additional data (unpublished) accumulated since our last published report, 2 show that Indian female

6 February 1973 LIVER PHYSIOLOGY AND DISEASE 303 patients with cholesterol gallstones have significantly increased cholesterol secretion when compared with Indian females without gallstones. Metzger and Grundy, 21 using physiological method for the determination of hourly secretion rates of biliary lipids,22 have also demonstrated that Indian females with cholesterol gallstones have a significant increase in daily cholesterol secretion in view of the decrease in bile acid pool size. 2 In contrast, patients with cirrhosis have a decrease in both cholesterol secretion and the bile acid pool size. The bile acid pool is reduced in these patients 11 because of an impairment in the synthesis of cholic acid. Chenodeoxycholic acid synthesis is virtually unaffected. The marked decrease in cholesterol secretion in cirrhotic patients could be due to an impaired synthesis of liver cholesterol or to a defect in the transport of cholesterol across the canalicular membrane during its incorporation into the biliary micelle. Additional studies are needed to delineate the factors regulating synthesis and secretion of biliary cholesterol in patients with liver disease. REFERENCES 1. Vlahcevic ZR, Bell CC Jr, Buhac I, et al: Diminished bile acid pool size in patients with gallstones. Gastroenterology 59: , Vlahcevic ZR, Bell CC Jr, Gregory DH, et al: Relationship of bile acid pool size to the formation of lithogenic bile in female Indians of the Southwest. Gastroenterology 62:73-83, Bell CC Jr, McCormick WC III, Gregory DH, et al: Relationship of bile acid pool size to the formation of lithogenous bile in male Indians of the Southwest. Surg Gynecol Obstet 134: , Swell Leon, Bell CC Jr, Vlahcevic ZR: Relationship of bile acid pool size to biliary lipid excretion and the formation of lithogenic bile in man. Gastroenterology 61: , Heaton KW, Read AE: Gallstones in patients with disorders of the terminal ileum and disturbed bile salt metabolism. B Med J 3: , Cohen S, Kaplan M, Gottlieb L, et al: Liver disease and gallstones in regional enteritis. Gastroenterology 60: , Vlahcevic ZR, Buhac I, Farrar JT, et al: Bile acid metabolism in patients with cirrhosis. 1. Kinetic aspects of cholic acid metabolism. Gastroenterology 60: , Bouchier lad: Postmortem study of the frequency of gallstones in patients with cirrhosis of the liver. Gut 10: , Nicholas P, Rinaudo PA, Conn HO: Increased incidence of cholelithiasis in Laennec's cirrhosis. A postmortem evaluation of pathogenesis. Gastroenterology 63: , Vlahcevic ZR, Miller JR, Farrar JT, et al: Kinetics and pool size of primary bile acids in man. Gastroenterology 61:85-90, Vlahcevic ZR, Juttijudata P, Bell CC Jr, et al: Bile acid metabolism in patients with cirrhosis. II. Cholic and chenodeoxycholic acid metabolism. Gastroenterology 62: , Folch J, Lees M, Sloane-Stanley HG: A simple method for the isolation and purification of total lipids from animal tissues. J BioI Chern 226: , Bartlett GR: Phosphorus assay in column chromatography. J BioI Chern 234: , Sperry W, Webb M: Revision of Schoenheimer Sperry method for cholesterol determination. J BioI Chern 187:97-102, Admirand WH, Small DM: The physicochemical basis of cholesterol gallstone formation in man. J Clin Invest 47: , Swell L, Entenman C, Leong GF, et al: Bile acids and lipid metabolism. IV. Influence of bile acids on biliary and liver organelle phospholipids and cholesterol. Am J Physiol 215: , Swell L, Bell CC Jr, Entenman C: Bile acids and lipid metabolism. III. Influence of bile acids on phospholipids in liver and bile of the isolated perfused dog liver. Biochim Biophys Acta 164: , Bell CC Jr, Vlahcevic ZR, Swell L: Alterations in the lipids of human hepatic bile after the oral administration of bile salts. Surg Gynecol Obstet 132:36-42, Nilsson S, Schersten T: Importance of bile acids for phospholipid secretion into human hepatic bile. Gastroenterology 57: , Balint JA, Beeler DA, Kyriakides EC, et al: The effect of bile salts upon lecithin synthesis. J Lab Clin Med 77: , Metzger AL, Adler R, Grundy SM: Role of biliary cholesterol in production oflithogenic bile (abstr). Gastroenterology 62:855, Grundy SM, Metzger AL: A physiological method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology 62: , 1972