Serum Bile Acids as Related to Bile Acid Secretion in Liver Disease ELDON A. SHAFFER, MD, and E L L E N R. GORDON, PhD
The level of serum bile acids and their rate of secretion into the duodenum were measured in normal subjects and patients with cholesterol gallstones and hepatobiliary disease. These studies were undertaken to determine if changes in serum bile acids reflected alterations in bile acid secretion within the enterohepatic circulation, especially in association with disordered bile acid metabolism and liver dysfunction. To quantitate bile acid secretion, an intestinal marker-perfusion technique was used which measured duodenal output under stimulated but steady-state conditions. During these perfusion studies, serum bile acids were determined at hourly intervals. In normal subjects, serum bile acids during fasting were low (6.6 • 0.7 tzM) with only a modest rise occurring after a fat meal (7.6 +- 0.7 lzM). The average bile acid secretion rate in these subjects was 1321 tzmol/hr, and little fluctuation was noted in the serum levels (7,5 + 0.8 ixM) during the perfusion studies. Patients with cholesterol gallstones demonstrated a decreased rate of bile acid secretion at 714 tzmol/hr but had normal serum values. After cholecystectomy, their bile acid secretion rate increased without any corresponding change in serum levels. In patients with liver disease, however, serum bile acids were elevated while bile acid secretion was reduced. Thus, in normal subjects the liver is quite capable of handling an increased endogenous bile acid return with only a small rise in serum levels. In liver disease, bile acid secretion is reduced while serum concentrations are increased, implying a defect in bile acid handling by the liver. In cholesterol gallstone disease, serum levels do not reflect the abnormality in bile acid metabolism. The relation between bile acid concentration in the serum and secretion rate into the enterohepatic circulation is therefore complex, depending on whether or not hepatic dysfunction is present or if bile acid metabolism is abnormal.
Bile acid secretion and metabolism are closely intertwined in the liver. Not only does the liver efficiently clear bile acids from the systemic circulation and secrete them into bile, but also the liver is the exclusive site for bile acid synthesis. Because bile acids represent a major excretory load for the liver, Carey logically reasoned that serum concentrations of bile acids should reflect any deterioration in From the McGill University Medical Clinic and the Division of Gastroenterology, The Montreal General Hospital, Montreal, Quebec, Canada. This work was supported by grant MA-5080 from the Medical Research Council of Canada. Address for reprint requests: Dr. Eldon A. Shaffer, Gastrointestinal Research Unit, Division of Medicine, Faculty of Medicine, University of Calgary, Calgary T2N 1N4, Alberta, Canada.
392
hepatobiliary function (1). Initially, this concept could not be examined because the early methodology used was too insensitive to accurately measure the normally low range of serum bile acids (2, 3). The recent development of sensitive assay methods has changed this and enabled the investigator to use the serum bile acids as a subtle index of hepatobiliary function (4). In addition, eating, which causes gallbladder contraction and more rapid intestinal transit, stimulates the enterohepatic circulation and increases the endogenous bile acid return to the liver. Thus increased 2-hr postprandial values have become a refined manifestation of minimal liver disease (5). This further led to the development of a bile acid tolerance test in which p l a s m a disDigestive Diseases, Vol. 23, No. 5 (May 1978)
0002-9211/78/0500-0392505.00/1@ 1978DigestiveDisease Systems, Inc.
SERUM BILE ACIDS a p p e a r a n c e curves were m e a s u r e d following an exogenous load of bile acid (6, 8). The m e c h a n i s m underlying this increase in serum bile acids, h o w e v e r , has not been elucidated. Such an increment associated with liver disease could result f r o m defective u p t a k e or handling by the liver, or f r o m altered biliary secretion into the d u o d e n u m . Previous studies h a v e a s s u m e d that liver disease is associated with disturbances in the enterohepatic circulation, such as reduced bile acid secretion and hepatic transport, e v e n though none h a v e actually m e a s u r e d both secretion into the enterohepatic circulation and the resultant effect on serum bile acids. A good example of a subtle liver defect w h e r e bile acid metabolism is disturbed is cholesterol gallstone disease. H e r e a small bile acid pool is associated with a reduced secretion rate (9) and a slight inefficiency in intestinal absorption (10). This situation p r e s u m a b l y results in a decreased hepatic return which, in the a b s e n c e of overt liver disease, should be associated with normal or low s e r u m levels. U n e x p e c t e d l y , Bell and co-workers (11) found increased values in fasting patients with cholesterol gallstones who did not have any evidence of biliary obstruction. U n d e r these circumstances, then, the p r e s e n c e of elevated s e r u m bile acids would suggest that hepatic extraction from portal blood and secretion into bile m a y be the abnormality in cholesterol gallstone disease. A p r i m a r y transport defect of bile acids across the h e p a t o c y t e might result in diminished bite acid secretion with a resultant increase in biliary cholesterol saturation. If hepatic return exceeded secretion, bile acids could accumulate in the liver, inhibit synthesis, and so reduce the bile acid pool, To further define this relationship, we have simultaneously m e a s u r e d bile acid output into the small intestine and serum bile acid concentrations. Healthy subjects were c o m p a r e d to two groups with abnormal bile acid m e t a b o l i s m - - p a t i e n t s with cholesterol gallstones and patients with hepatobiliary disease.
liver disease. Fatty infiltration was present in seven patients with morbid obesity (being almost twice their ideal body weight). Liver biopsies were performed as part of their p r e o p e r a t i v e evaluation for ileojejunal bypass. Stable cirrhosis was documented clinically in eight alcoholic patients and confirmed morphologically in six. Finally, marked cholestasis was present in four patients with cholangiolitic hepatitis, alcoholic hepatitis, and in two instances, large duct obstruction. Cases were carefully documented, including routine liver function tests (serum bilirubin, alkaline phosphatase, transaminase, and protein) and where appropriate, cholangiography. The patients with cholesterol gallstones and the obese patients had gallbladders which functioned normally on oral cholecystography. The routine laboratory tests were all normal in the control subjects, the patients with cholesterol gallstones and also the obese patients with fatty infiltration. The cirrhotic group, although anicteric, all demonstrated some abnormalities, but were clinically and biochemically stable, having abstained from alcohol for over 10 days. Those with cholestasis were distinctly jaundiced. The patient groups studied were adult Caucasians (23-87 years), within 12% of their ideal body weight* except for the obese patients, and nearly equally divided as to sex, although the cirrhotics were all men. Verbal and written consent was obtained from every subject.
MATERIALS AND METHODS
Serum Bile Acids Serum bile acid concentrations were measured by the fluorometric 3c~-hydroxysteroid dehydrogenase method (17) as modified by Barnes et al (18). Serum levels
Subjects Studies were performed on 14 control subjects without any evidence of hepatobiliary disease. A group of patients with cholesterol gallstones but without biliary obstruction were similarly examined. Five were studied before, and 15 at least one month after, full recovery from cholecystectomy. Four of these were studied both before and after surgery. These two groups without overt liver disease were compared to three other groups with documented Digestive Diseases, Vol. 23, No. 5 (May 1978)
Perfusion Studies Bile acid secretion from the liver into the enterohepatic circulation was measured by a duodenal marker-perfusion technique (12) which we have used to measure bilirubin (13) and biliary lipid secretion (9). Briefly, fasting subjects were constantly perfused through a poiyvinyl tube with an essential amino acid solution~ (14) which tonically contracts the gallbladder and provides a source of calories during the study. This functionally eliminated the gallbladder so that under steady-state conditions duodenal output represented hepatic secretion (12). Perfusion studies were carried out over 10 hr, and the hourly rate of bile acid secretion into the duodenum was determined by a constant infusion of bromosulfophthalein (BSP), in accordance with marker dilution principles (15). After an equilibrium period of 4-6 hr, bile salt output into the duodenum became quite constant, indicating the establishment of steady-state conditions of hepatic secretion. Only the last 6 hr of steady secretion were therefore considered. Duodenal bile acid concentration was quantitated by the 3~-hydroxysteroid dehydrogenase assay, as previously described (9), while BSP was assayed colorimetricatly (t6).
*Metropolitian Life Insurance Company. New weight standards for men and women. Statistical Bulletin 40. New York, 1959. ?5% protein hydrolysate in 5% glucose ("aminosol" Abbott Laboratories, Montreal, Canada) with added L-valine (25.6 mM), Lmethionine (40.2 mM), and L-phenylalamine (18.2 raM) Eastman Kodak Co., Rochester, New York.
393
SHAFFER
AND
GORDON
TABLE 1. EFFECT OF A MEAL ON SERUM BILE ACIDS
Serum bile acids
(t~)* Disease state
Subjects
Fasting
None Cholesterol gallstones Before ch01ecystectomy Aftercholecystectomy Fatty liver of morbid obesity Cirrhosis Cholestasis
28
6.6 -
0.7
5 15 8 17 6
7.7 • 11.1 • 16.9 --34.2--81.2 •
1.4 1.2 6.2 6.0 28.0
Postprandial 7.6 -+ 0.7 10.1 9.6 26.2 52.1 74.9
--- 3.1 • i.1 _+ 5,3 • 7.3t ___ 25.9
*Mean (--- SEM) serum bile acid concentrations while fasting and 2 hr after a meal. tPaired t test indicates that postprandial levels differ significantly from fasting level (P < 0.005).
were determined in the fasting basal state and then at hourly intervals during the perfusion studies. In addition, serum concentrations were quantitated after an overnight fast and 2 hr after a standard 500-kcal breakfast (18). Fourteen additional subjects without hepatobiliary disease and 12 other patients with liver disease had serum bile acid levels measured while fasting and 2 hr after the test meal. Statistical significance of the data was evaluated by the Student's t test for paired or unpaired data where applicable. RESULTS
The average serum bile acid concentration was 6.6 - 0.7/zM in fasting subjects without hepatobiliary disease (Table 1), while the upper limit of normal was 15/zM (18). The individual values were not affected by sex or age, and only a modest increase was found after meals. Patients with cholesterol gallstones without biliary obstruction had normal fasting serum bile acid levels, and a slight but insignificant (P > 0.5) increase was observed 2 hr postprandially. After removal of the gallbladder, fasting values were significantly (P < 0.005) greater than that of the control subjects, but remained with~n normal limits and no postprandial rise occurred. The eight morbidly obese patients whose liver biopsies showed fatty infiltration had serum bile acid concentrations significantly (P < 0.00l) higher than normal. This obese group showed a postprandial rise, although the increase did not quite reach significance (P < 0.1). Serum levels, however, did not correlate with the degree of morphological fatty change. Indeed, four patients with moderately severe fatty infiltration had normal fasting values, althoUgh in two the postprandial levels were abnormal. In the patients with cirrhosis,
394
serum bile acids were markedly elevated while fasting and rose significantly (P < 0.005) following a meal. The highest values were observed in the fasting patients with cholestasis, but there was no further rise in the postprandial state. During the perfusion studies when steady-state conditions of bile acid secretion into the duodenum were established, serum bile acid levels remained constant (Figure 1). It was therefore possible to compare these serum levels with the rate of entry of bile acids into the duodenum (Table 2). The low serum bile acid concentrations in the control subjects were associated with a biliary secretion rate of 1321 tzmol/hr. The patients with cholesterol gallstones also demonstrated low serum concentrations, even though duodenal secretion into the enterohepatic circulation was significantly (P < 0.05) reduced. Following removal of the gallbladder, the secretion rates of bile acids increased to the normal range, but this was not associated with any change in serum bile acid concentration (Figure 2). The patients with fatty liver of morbid obesity had duodenal secretion rates similar to those in the control group, whereas the corresponding serum bile acids were significantly (P < 0.01) greater than normal. In both cirrhosis and cholestasis, serum bile acids were distinctly elevated and a s s o c i a t e d with a marked decrease in duodenal secretion. Serum bile acids in these patients with liver disease showed an inverse correlation with duodenal bile acid secretion (Figure 3). As bile acid secretion rates decreased, serum bile acid concentrations increased. The exception to this was patients with cholesterol gallstones. When the gallbladder harboring the stones was intact, bile acid secretion was reduced but serum levels were normal. After cholecystecDigestive Diseases, Vol. 23, No. 5 (May 1978)
SERUM BILE ACIDS 100
SERUM
%xx
80
xx~
~o. . . . o~ ~
DISCUSSION o~
J'~ o
BILE ACID 60 CONCENTRATION
p moles/L
402O
2000
9
BILE ACID 1.500
9
SECRETION ~urnoles/h
A
10009
- .....
9 . . . . . . .
. .......
2----
7
750-
TIME-hours
Fig 1. Hourly serum bile acid concentrations and the corresponding bile acid secretion rate in two representative duodenal perfusion studies. Serum bile acid levels (shown as open symbols) obtained each hour were compared to the rate of bile acid secretion into the duodenum (shown as closed symbols) during the hour. A typical subject without any hepatobiliary disease (represented as triangles) demonstrated rather low, fiat serum levels despite the high bile acid secretion in the first few hours, probably the result of gallbladder contraction. Thereafter secretion became steady. By contrast, a patient with cirrhosis (represented as circles) had a very high serum bile acid concentration despite a continuously reduced secretory rate.
tomy, serum concentrations remained low although the secretion rate rose to the values seen in the control group (Table 2).
Bile acids are normally contained within the enterohepatic circulation, being secreted by the liver, absorbed mainly by the ileum, and returned to the liver by the portal vein. The superb efficiency of intestinal absorption and hepatic extraction of bile acids results in their conservation. In fact, about 97% of bile acids that enter the intestine are absorbed, largely by an active transport system in the distal small bowel, while more than 95% of the total bile acids returning via the portal vein are actively cleared by the liver and resecreted into bile (19). This remarkable arrangement normally keeps fecal losses small, and allows only minute quantities of bile acids to appear in the systemic circulation. The concentration of bile acids in the systemic circulation therefore represents the instantaneous balance between input from intestinal absorption and uptake by the liver. The liver is normally able to rapidly clear bile acids so that serum levels increase only transiently after a meal, presumably due to a spillover from the portal to the s y s t e m i c circulation (20). In the presence of normal liver function then, serum levels are probably directly related to secretion into the intestine and subsequent absorption. If liver function is abnormal, alterations in this dynamic system become complex but should be reflected by changes in serum values. To quantitate bile acid secretion within the enterohepatic circulation, an intestinal marker-perfusion technique was used which measured secretion un-
TABLE 2. SERUM BILE ACID CONCENTRATIONS DURING STEADY-STATE BILE ACID SECRETION INTO THE DUODENUM Bile acids* Disease state
Subject
Serum levels (ixM)
Duodenal secretion ( ixmol/hr)
None Cholesterol gallstones Beforecholecystectomy After cholecystectomy Fatty liver of morbid obesity Cirrhosis Cholestasis
14
7.5 _+ 0.8
1321 _+ 126
5 15 7 8 4
8.6 8.7 22.8 45.8 85.0
_+ 1.4 _+ 0.9 _+_ 5.3t _+ 8.5? _+ 39.7t
714 1411 1029 859 488
_+ 148t _+ 103 _+ 118 _+ 154t _ 144t
*Mean (_+ SEM) hourly bile acid concentration in the serum (/xM) and the bile acid secretion into the duodenum (/xmol/hr) during the perfusion study. tGroup t test comparison showed a significant (P < 0.05) difference from normal subjects without hepatobiliary disease. Digestive Diseases, Vol. 23, No. 5 (May 1978)
395
SHAFFER AND GORDON
2000] BILEACID 1500"~ SECRETION -~ u rnoles/hr 10001
SERUM 2O7
B,LEAC,DS -t )JM
O_I
PRE POST CHOLECYSTECTOMY
s~176 t O.j
PRE POST CHOLECYSTECTOMY
Fig 2. T h e effect of c h o l e c y s t e c t o m y on s e r u m bile acid concentrations in contrast to the bile acid secretion rate. Individual data, plotted for 4 patients with cholesterol gallstones, are connected for the respective perfusion studies done before and after cholecystectomy. Preoperative serum concentrations were low in association with a reduced secretion rate. After surgery, s e r u m levels r e m a i n e d low despite the increase in bile acid secretion.
der stimulated but steady-state conditions (9, 12). The advantage of establishing such a steady state is that hepatic secretion into the duodenum can be directly related to bile acid concentration in the serum. Use of an alternative marker-perfusion system, which introduces three test meals to simulate the effects of fasting and feeding (21), would have created problems in defining the time lag between events measured in the duodenum and any resultant changes in the serum. Furthermore, duodenal output under these conditions represents the algebraic sum of hepatic secretion plus gallbladder filling or emptying, so that hepatic secretion is not being measured and cannot be properly related to serum c o n c e n t r a t i o n s . Although these two perfusion methods have yielded different results in studies on cholesterol gallstone disease (9, 21), their estimates of 24-hr outputs agree rather well (22). The present study has correlated serum bile acids with the bile acid secretion rate in order to define the effect of disordered hepatic function. In our normal subjects, the liver was quite capable of handling an increased endogenous bile acid return. Even under conditions in which the enterohepatic circulation was stimulated either by a meal or amino acid infusion, only a small rise in bile acids occurred. A direct relationship between intestinal secretion and serum bile acids could not be detected in this nor-
396
mal group, perhaps because the fluorometric enzyme assay used lacked the sensitivity necessary to detect subtle changes at such low normal serum concentrations. In the patients with liver disease, bile acid concentrations within the systemic circulation were elevated while bile acid secretion into the small intestine was reduced. The basis for this inverse relation between serum concentration and biliary secretion is a hepatic transport defect. More severe impairment of liver function results in higher serum concentrations but lower rates of bile acid secretion. Such inefficient bile acid clearance could result from impaired hepatic u p t a k e , vascular shunting, or reduced secretion with back-diffusion into the serum. Serum values, however, were not always elevated in the presence of morphological alterations in the liver. This has been noted previously (23), and may merely indicate that either such assays are not infallible in detecting liver disease or morphological change does not necessarily mean dysfunction. In fact, serum bile acids are not only dependent on the capacity of the hepatic transport system but also on the rate of hepatic return within the enterohepatic circulation. During fasting, serum levels are reduced even in some patients with liver disease because bile acid secretion into the intestine is decreased although not abolished by gallbladder filling (24). If bile acid malabsorption were
120100SERUM80-
BILE ACIDS 60.,um/L 40i
20-
,11
T
; I
s6o
~obo
9
I
1so6
BILE ACID SECRETION - ,,umoles/L Fig 3. The relationship b e t w e e n s e r u m bile acid concentration and the corresponding bile acid secretion rate. Normal controls are compared to the patient groups with liver disease: fatty liver of morbid obesity, alcoholic cirrhosis, and cholestasis. M e a n (~ SEM)i s g i v e n for each group. S e r u m levels s h o w e d an inverse correlation with bile acid secretion. Digestive Diseases, Vol. 23, No. 5 (May 1978)
SERUM BILE ACIDS
present, only modest or no serum elevation would be expected. The relationship between bile acid secretion from the liver and the corresponding serum concentrations is clearly altered in cholesterol gallstone disease. Here, a low rate of bile acid secretion, the product of a small circulating bile acid pool (9), is associated with normal serum values. When bile acid secretion is increased in these patients after removal of the gallbladder, serum concentrations remain virtually unchanged. This suggests that hepatic uptake in cholesterol gallstone disease is normal, although a very subtle defect in bile acid flux through the liver cannot be completely eliminated. The high normal serum values present during fasting likely results from a continuous secretion of bile acids into the intestine after cholecystectomy. In contrast to the normal serum bile acids in our patients, the elevated values found by Bell et al (11) may indicate the inclusion of gallstone patients, who can occasionally exhibit biochemical and morphological abnormalities (25). Hence, changes in serum levels reflect the ability of the liver to transport bile acids, but unfortunately do not reveal the abnormality in bile acid metabolism which is associated with cholesterol gallstone disease. ACKNOWLEDGMENTS W e w i s h to t h a n k M s R. O ' B r i e n , M s A. T u o h y , a n d M s K. S a m o d a i for t h e i r e x p e r t t e c h n i c a l a n a l y s i s , M s S. S c h u h a r t for typing this m a n u s c r i p t , a n d Drs. S. B a r n e s a n d U. F a r a c k for t h e i r a d v i c e a n d a s s i s t a n c e .
REFERENCES 1. Carey JB Jr: The serum trihydroxy-dihydroxy bile acid ratio in liver and biliary tract disease. J Clin Invest 37:1494-1503, 1958 2. Sherlock S, Walshe V: Blood cholates in normal subjects and in liver disease. Clin Sci 6:223-234, 1948 3. Osborn EC, Wooton IDP, daSilva LC, Sherlock S. Serumbile-acid levels in liver disease. Lancet 2:1049-1053, 1959 4. Editorial: Serum bile acids as a test of liver function. Lancet 2:267, 1975 5. Kaplowitz N, Kok E, Javitt NB: Postprandial serum bile acid for the d e t e c t i o n of h e p a t o b i l i a r y disease. J A M A 225:292-293, 1973 6. Kaye MD, Struthers TE, Tidball JS, DeNiro E, Kern F Jr: Factors affecting plasma clearance of p4C]cholic acid in patients with cirrhosis. Clin Sci Mol Med 45:147-161, 1973 7. Cowen AE, Korman MG, Hofmann AF, Thomas PJ: Plasma disappearance of radioactivity after intravenous injection of
Digestive Diseases, Vol. 23, No. 5 (May 1978)
labeled bile acids in man. Gastroenterology 68:1567-1573, 1975 8. Korman, MG, LaRusso NF, Hoffman NE, Hofmann AF: Development of an intravenous bile acid tolerance test. Plasma disappearance of cholylglycine in health. N Engl J Med 292:1205-1209, 1975 9. Shaffer EA, Small DM: Biliary lipid secretion in cholesterol gallstone disease: The effect of cholecystectomy and obesity. J Clin Invest 59:828-840, 1977 10. Grundy SM, Metzger AL, Adler RD: Mechanisms of lithogenic bile formation in American Indian women with cholesterol gallstones. J Clin Invest 51:3026-3043, 1972 11. Bell GD, Mok HYL, Thwe M, Murphy GM, Henry K, Dowling RH: Liver structure and function in cholelithiasis: Effect of chenodeoxycholic acid. Gut 15:165-172, 1974 12. Grundy SM, Metzger AL: A physiologic method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology 62:1200-1217, 1972 13. Gordon ER, Shaffer EA, Sass-Kortsak A: Bilimbin secretion and conjugation in the Crigler-Najjar syndrome type II. Gastroenterology 70:761-765, 1976 14. Go VLW, Hofmann AF, Summerskill WHJ: Pancreozymin bioassay in man based on pancreatic enzyme secretion: Potency of specific amino acids and other digestive products. J Clin Invest 49:1558-1564, 1970 15. Fordtran JS: Marker perfusion techniques for measuring intestinal absorption in man. Gastroenterology 51:1089-1093, 1966 16. Seligson D, Marino J, Dodson E: Determination of sulfobromophthalein in serum. Clin Chem 3:638-645, 1957 17. Murphy GM, Billing BH, Baron DN: A fluorimetric and enzymatic method for the estimation of serum total bile acids. J Clin Pathol 123:594-598, 1970 18. Barnes S, Gallo GA, Trash DB, Morris JS: Diagnostic value of serum bile acid estimations in liver disease. J Clin Pathol 28:506-509, 1975 19. Small DM, Dowling RH, Redinger RN: The enterohepatic circulation of bile salts. Arch Intern Med 130:552-573, 1972 20. LaRusso NE, Korman MG, Hofmann NE, Hofmann AF: Dynamics of the enterohepatic circulation of bile acids: Postprandial serum concentrations of conjugates of cholic acid in health, cholecystectomized patients, and patients with bile acid malabsorption. N Engl J Med 291:689-692, 1974 21. Northfield TC, Hofmann AF: Biliary lipid output during three meals and an overnight fast. I Relationship to bile acid pool size and cholesterol saturation of bile in gallstone and control subjects. Gut 16:1-17, 1975 22. Mok HYI, yon Bergmann K, Grundy SM: Effects of intermittent versus continuous feeding on biliary lipid secretion in man. Gastroenterology 71:921, 1976 (abstract) 23. Milstein H J, Bloomer JR, Klatskin G: Serum bile acids in alcoholic liver disease. Am J Dig Dis 21:281-285, 1976 24. Shaffer EA, Beaudoin M, Small DM, O'Brien J, Williams L: Relationship between gallbladder function and the enterohepatic circulation of bile salts during fasting. Gastroenterology 66:775, 1974 25. Fromm H, Holz-Slomczyk M, Zobl H, Schmidt E, Schmidt FW: Studies of liver function and structure in patients with gallstones before and during treatment with chenodeoxycholic acid. Acta Hepato-Gastroenterol 22:359-369, 1975
397
The level of serum bile acids and their rate of secretion into the duodenum were measured in normal subjects and patients with cholesterol gallstones and hepatobiliary disease. These studies were undertaken to determine if changes in serum bile acids reflected alterations in bile acid secretion within the enterohepatic circulation, especially in association with disordered bile acid metabolism and liver dysfunction. To quantitate bile acid secretion, an intestinal marker-perfusion technique was used which measured duodenal output under stimulated but steady-state conditions. During these perfusion studies, serum bile acids were determined at hourly intervals. In normal subjects, serum bile acids during fasting were low (6.6 • 0.7 tzM) with only a modest rise occurring after a fat meal (7.6 +- 0.7 lzM). The average bile acid secretion rate in these subjects was 1321 tzmol/hr, and little fluctuation was noted in the serum levels (7,5 + 0.8 ixM) during the perfusion studies. Patients with cholesterol gallstones demonstrated a decreased rate of bile acid secretion at 714 tzmol/hr but had normal serum values. After cholecystectomy, their bile acid secretion rate increased without any corresponding change in serum levels. In patients with liver disease, however, serum bile acids were elevated while bile acid secretion was reduced. Thus, in normal subjects the liver is quite capable of handling an increased endogenous bile acid return with only a small rise in serum levels. In liver disease, bile acid secretion is reduced while serum concentrations are increased, implying a defect in bile acid handling by the liver. In cholesterol gallstone disease, serum levels do not reflect the abnormality in bile acid metabolism. The relation between bile acid concentration in the serum and secretion rate into the enterohepatic circulation is therefore complex, depending on whether or not hepatic dysfunction is present or if bile acid metabolism is abnormal.
Bile acid secretion and metabolism are closely intertwined in the liver. Not only does the liver efficiently clear bile acids from the systemic circulation and secrete them into bile, but also the liver is the exclusive site for bile acid synthesis. Because bile acids represent a major excretory load for the liver, Carey logically reasoned that serum concentrations of bile acids should reflect any deterioration in From the McGill University Medical Clinic and the Division of Gastroenterology, The Montreal General Hospital, Montreal, Quebec, Canada. This work was supported by grant MA-5080 from the Medical Research Council of Canada. Address for reprint requests: Dr. Eldon A. Shaffer, Gastrointestinal Research Unit, Division of Medicine, Faculty of Medicine, University of Calgary, Calgary T2N 1N4, Alberta, Canada.
392
hepatobiliary function (1). Initially, this concept could not be examined because the early methodology used was too insensitive to accurately measure the normally low range of serum bile acids (2, 3). The recent development of sensitive assay methods has changed this and enabled the investigator to use the serum bile acids as a subtle index of hepatobiliary function (4). In addition, eating, which causes gallbladder contraction and more rapid intestinal transit, stimulates the enterohepatic circulation and increases the endogenous bile acid return to the liver. Thus increased 2-hr postprandial values have become a refined manifestation of minimal liver disease (5). This further led to the development of a bile acid tolerance test in which p l a s m a disDigestive Diseases, Vol. 23, No. 5 (May 1978)
0002-9211/78/0500-0392505.00/1@ 1978DigestiveDisease Systems, Inc.
SERUM BILE ACIDS a p p e a r a n c e curves were m e a s u r e d following an exogenous load of bile acid (6, 8). The m e c h a n i s m underlying this increase in serum bile acids, h o w e v e r , has not been elucidated. Such an increment associated with liver disease could result f r o m defective u p t a k e or handling by the liver, or f r o m altered biliary secretion into the d u o d e n u m . Previous studies h a v e a s s u m e d that liver disease is associated with disturbances in the enterohepatic circulation, such as reduced bile acid secretion and hepatic transport, e v e n though none h a v e actually m e a s u r e d both secretion into the enterohepatic circulation and the resultant effect on serum bile acids. A good example of a subtle liver defect w h e r e bile acid metabolism is disturbed is cholesterol gallstone disease. H e r e a small bile acid pool is associated with a reduced secretion rate (9) and a slight inefficiency in intestinal absorption (10). This situation p r e s u m a b l y results in a decreased hepatic return which, in the a b s e n c e of overt liver disease, should be associated with normal or low s e r u m levels. U n e x p e c t e d l y , Bell and co-workers (11) found increased values in fasting patients with cholesterol gallstones who did not have any evidence of biliary obstruction. U n d e r these circumstances, then, the p r e s e n c e of elevated s e r u m bile acids would suggest that hepatic extraction from portal blood and secretion into bile m a y be the abnormality in cholesterol gallstone disease. A p r i m a r y transport defect of bile acids across the h e p a t o c y t e might result in diminished bite acid secretion with a resultant increase in biliary cholesterol saturation. If hepatic return exceeded secretion, bile acids could accumulate in the liver, inhibit synthesis, and so reduce the bile acid pool, To further define this relationship, we have simultaneously m e a s u r e d bile acid output into the small intestine and serum bile acid concentrations. Healthy subjects were c o m p a r e d to two groups with abnormal bile acid m e t a b o l i s m - - p a t i e n t s with cholesterol gallstones and patients with hepatobiliary disease.
liver disease. Fatty infiltration was present in seven patients with morbid obesity (being almost twice their ideal body weight). Liver biopsies were performed as part of their p r e o p e r a t i v e evaluation for ileojejunal bypass. Stable cirrhosis was documented clinically in eight alcoholic patients and confirmed morphologically in six. Finally, marked cholestasis was present in four patients with cholangiolitic hepatitis, alcoholic hepatitis, and in two instances, large duct obstruction. Cases were carefully documented, including routine liver function tests (serum bilirubin, alkaline phosphatase, transaminase, and protein) and where appropriate, cholangiography. The patients with cholesterol gallstones and the obese patients had gallbladders which functioned normally on oral cholecystography. The routine laboratory tests were all normal in the control subjects, the patients with cholesterol gallstones and also the obese patients with fatty infiltration. The cirrhotic group, although anicteric, all demonstrated some abnormalities, but were clinically and biochemically stable, having abstained from alcohol for over 10 days. Those with cholestasis were distinctly jaundiced. The patient groups studied were adult Caucasians (23-87 years), within 12% of their ideal body weight* except for the obese patients, and nearly equally divided as to sex, although the cirrhotics were all men. Verbal and written consent was obtained from every subject.
MATERIALS AND METHODS
Serum Bile Acids Serum bile acid concentrations were measured by the fluorometric 3c~-hydroxysteroid dehydrogenase method (17) as modified by Barnes et al (18). Serum levels
Subjects Studies were performed on 14 control subjects without any evidence of hepatobiliary disease. A group of patients with cholesterol gallstones but without biliary obstruction were similarly examined. Five were studied before, and 15 at least one month after, full recovery from cholecystectomy. Four of these were studied both before and after surgery. These two groups without overt liver disease were compared to three other groups with documented Digestive Diseases, Vol. 23, No. 5 (May 1978)
Perfusion Studies Bile acid secretion from the liver into the enterohepatic circulation was measured by a duodenal marker-perfusion technique (12) which we have used to measure bilirubin (13) and biliary lipid secretion (9). Briefly, fasting subjects were constantly perfused through a poiyvinyl tube with an essential amino acid solution~ (14) which tonically contracts the gallbladder and provides a source of calories during the study. This functionally eliminated the gallbladder so that under steady-state conditions duodenal output represented hepatic secretion (12). Perfusion studies were carried out over 10 hr, and the hourly rate of bile acid secretion into the duodenum was determined by a constant infusion of bromosulfophthalein (BSP), in accordance with marker dilution principles (15). After an equilibrium period of 4-6 hr, bile salt output into the duodenum became quite constant, indicating the establishment of steady-state conditions of hepatic secretion. Only the last 6 hr of steady secretion were therefore considered. Duodenal bile acid concentration was quantitated by the 3~-hydroxysteroid dehydrogenase assay, as previously described (9), while BSP was assayed colorimetricatly (t6).
*Metropolitian Life Insurance Company. New weight standards for men and women. Statistical Bulletin 40. New York, 1959. ?5% protein hydrolysate in 5% glucose ("aminosol" Abbott Laboratories, Montreal, Canada) with added L-valine (25.6 mM), Lmethionine (40.2 mM), and L-phenylalamine (18.2 raM) Eastman Kodak Co., Rochester, New York.
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TABLE 1. EFFECT OF A MEAL ON SERUM BILE ACIDS
Serum bile acids
(t~)* Disease state
Subjects
Fasting
None Cholesterol gallstones Before ch01ecystectomy Aftercholecystectomy Fatty liver of morbid obesity Cirrhosis Cholestasis
28
6.6 -
0.7
5 15 8 17 6
7.7 • 11.1 • 16.9 --34.2--81.2 •
1.4 1.2 6.2 6.0 28.0
Postprandial 7.6 -+ 0.7 10.1 9.6 26.2 52.1 74.9
--- 3.1 • i.1 _+ 5,3 • 7.3t ___ 25.9
*Mean (--- SEM) serum bile acid concentrations while fasting and 2 hr after a meal. tPaired t test indicates that postprandial levels differ significantly from fasting level (P < 0.005).
were determined in the fasting basal state and then at hourly intervals during the perfusion studies. In addition, serum concentrations were quantitated after an overnight fast and 2 hr after a standard 500-kcal breakfast (18). Fourteen additional subjects without hepatobiliary disease and 12 other patients with liver disease had serum bile acid levels measured while fasting and 2 hr after the test meal. Statistical significance of the data was evaluated by the Student's t test for paired or unpaired data where applicable. RESULTS
The average serum bile acid concentration was 6.6 - 0.7/zM in fasting subjects without hepatobiliary disease (Table 1), while the upper limit of normal was 15/zM (18). The individual values were not affected by sex or age, and only a modest increase was found after meals. Patients with cholesterol gallstones without biliary obstruction had normal fasting serum bile acid levels, and a slight but insignificant (P > 0.5) increase was observed 2 hr postprandially. After removal of the gallbladder, fasting values were significantly (P < 0.005) greater than that of the control subjects, but remained with~n normal limits and no postprandial rise occurred. The eight morbidly obese patients whose liver biopsies showed fatty infiltration had serum bile acid concentrations significantly (P < 0.00l) higher than normal. This obese group showed a postprandial rise, although the increase did not quite reach significance (P < 0.1). Serum levels, however, did not correlate with the degree of morphological fatty change. Indeed, four patients with moderately severe fatty infiltration had normal fasting values, althoUgh in two the postprandial levels were abnormal. In the patients with cirrhosis,
394
serum bile acids were markedly elevated while fasting and rose significantly (P < 0.005) following a meal. The highest values were observed in the fasting patients with cholestasis, but there was no further rise in the postprandial state. During the perfusion studies when steady-state conditions of bile acid secretion into the duodenum were established, serum bile acid levels remained constant (Figure 1). It was therefore possible to compare these serum levels with the rate of entry of bile acids into the duodenum (Table 2). The low serum bile acid concentrations in the control subjects were associated with a biliary secretion rate of 1321 tzmol/hr. The patients with cholesterol gallstones also demonstrated low serum concentrations, even though duodenal secretion into the enterohepatic circulation was significantly (P < 0.05) reduced. Following removal of the gallbladder, the secretion rates of bile acids increased to the normal range, but this was not associated with any change in serum bile acid concentration (Figure 2). The patients with fatty liver of morbid obesity had duodenal secretion rates similar to those in the control group, whereas the corresponding serum bile acids were significantly (P < 0.01) greater than normal. In both cirrhosis and cholestasis, serum bile acids were distinctly elevated and a s s o c i a t e d with a marked decrease in duodenal secretion. Serum bile acids in these patients with liver disease showed an inverse correlation with duodenal bile acid secretion (Figure 3). As bile acid secretion rates decreased, serum bile acid concentrations increased. The exception to this was patients with cholesterol gallstones. When the gallbladder harboring the stones was intact, bile acid secretion was reduced but serum levels were normal. After cholecystecDigestive Diseases, Vol. 23, No. 5 (May 1978)
SERUM BILE ACIDS 100
SERUM
%xx
80
xx~
~o. . . . o~ ~
DISCUSSION o~
J'~ o
BILE ACID 60 CONCENTRATION
p moles/L
402O
2000
9
BILE ACID 1.500
9
SECRETION ~urnoles/h
A
10009
- .....
9 . . . . . . .
. .......
2----
7
750-
TIME-hours
Fig 1. Hourly serum bile acid concentrations and the corresponding bile acid secretion rate in two representative duodenal perfusion studies. Serum bile acid levels (shown as open symbols) obtained each hour were compared to the rate of bile acid secretion into the duodenum (shown as closed symbols) during the hour. A typical subject without any hepatobiliary disease (represented as triangles) demonstrated rather low, fiat serum levels despite the high bile acid secretion in the first few hours, probably the result of gallbladder contraction. Thereafter secretion became steady. By contrast, a patient with cirrhosis (represented as circles) had a very high serum bile acid concentration despite a continuously reduced secretory rate.
tomy, serum concentrations remained low although the secretion rate rose to the values seen in the control group (Table 2).
Bile acids are normally contained within the enterohepatic circulation, being secreted by the liver, absorbed mainly by the ileum, and returned to the liver by the portal vein. The superb efficiency of intestinal absorption and hepatic extraction of bile acids results in their conservation. In fact, about 97% of bile acids that enter the intestine are absorbed, largely by an active transport system in the distal small bowel, while more than 95% of the total bile acids returning via the portal vein are actively cleared by the liver and resecreted into bile (19). This remarkable arrangement normally keeps fecal losses small, and allows only minute quantities of bile acids to appear in the systemic circulation. The concentration of bile acids in the systemic circulation therefore represents the instantaneous balance between input from intestinal absorption and uptake by the liver. The liver is normally able to rapidly clear bile acids so that serum levels increase only transiently after a meal, presumably due to a spillover from the portal to the s y s t e m i c circulation (20). In the presence of normal liver function then, serum levels are probably directly related to secretion into the intestine and subsequent absorption. If liver function is abnormal, alterations in this dynamic system become complex but should be reflected by changes in serum values. To quantitate bile acid secretion within the enterohepatic circulation, an intestinal marker-perfusion technique was used which measured secretion un-
TABLE 2. SERUM BILE ACID CONCENTRATIONS DURING STEADY-STATE BILE ACID SECRETION INTO THE DUODENUM Bile acids* Disease state
Subject
Serum levels (ixM)
Duodenal secretion ( ixmol/hr)
None Cholesterol gallstones Beforecholecystectomy After cholecystectomy Fatty liver of morbid obesity Cirrhosis Cholestasis
14
7.5 _+ 0.8
1321 _+ 126
5 15 7 8 4
8.6 8.7 22.8 45.8 85.0
_+ 1.4 _+ 0.9 _+_ 5.3t _+ 8.5? _+ 39.7t
714 1411 1029 859 488
_+ 148t _+ 103 _+ 118 _+ 154t _ 144t
*Mean (_+ SEM) hourly bile acid concentration in the serum (/xM) and the bile acid secretion into the duodenum (/xmol/hr) during the perfusion study. tGroup t test comparison showed a significant (P < 0.05) difference from normal subjects without hepatobiliary disease. Digestive Diseases, Vol. 23, No. 5 (May 1978)
395
SHAFFER AND GORDON
2000] BILEACID 1500"~ SECRETION -~ u rnoles/hr 10001
SERUM 2O7
B,LEAC,DS -t )JM
O_I
PRE POST CHOLECYSTECTOMY
s~176 t O.j
PRE POST CHOLECYSTECTOMY
Fig 2. T h e effect of c h o l e c y s t e c t o m y on s e r u m bile acid concentrations in contrast to the bile acid secretion rate. Individual data, plotted for 4 patients with cholesterol gallstones, are connected for the respective perfusion studies done before and after cholecystectomy. Preoperative serum concentrations were low in association with a reduced secretion rate. After surgery, s e r u m levels r e m a i n e d low despite the increase in bile acid secretion.
der stimulated but steady-state conditions (9, 12). The advantage of establishing such a steady state is that hepatic secretion into the duodenum can be directly related to bile acid concentration in the serum. Use of an alternative marker-perfusion system, which introduces three test meals to simulate the effects of fasting and feeding (21), would have created problems in defining the time lag between events measured in the duodenum and any resultant changes in the serum. Furthermore, duodenal output under these conditions represents the algebraic sum of hepatic secretion plus gallbladder filling or emptying, so that hepatic secretion is not being measured and cannot be properly related to serum c o n c e n t r a t i o n s . Although these two perfusion methods have yielded different results in studies on cholesterol gallstone disease (9, 21), their estimates of 24-hr outputs agree rather well (22). The present study has correlated serum bile acids with the bile acid secretion rate in order to define the effect of disordered hepatic function. In our normal subjects, the liver was quite capable of handling an increased endogenous bile acid return. Even under conditions in which the enterohepatic circulation was stimulated either by a meal or amino acid infusion, only a small rise in bile acids occurred. A direct relationship between intestinal secretion and serum bile acids could not be detected in this nor-
396
mal group, perhaps because the fluorometric enzyme assay used lacked the sensitivity necessary to detect subtle changes at such low normal serum concentrations. In the patients with liver disease, bile acid concentrations within the systemic circulation were elevated while bile acid secretion into the small intestine was reduced. The basis for this inverse relation between serum concentration and biliary secretion is a hepatic transport defect. More severe impairment of liver function results in higher serum concentrations but lower rates of bile acid secretion. Such inefficient bile acid clearance could result from impaired hepatic u p t a k e , vascular shunting, or reduced secretion with back-diffusion into the serum. Serum values, however, were not always elevated in the presence of morphological alterations in the liver. This has been noted previously (23), and may merely indicate that either such assays are not infallible in detecting liver disease or morphological change does not necessarily mean dysfunction. In fact, serum bile acids are not only dependent on the capacity of the hepatic transport system but also on the rate of hepatic return within the enterohepatic circulation. During fasting, serum levels are reduced even in some patients with liver disease because bile acid secretion into the intestine is decreased although not abolished by gallbladder filling (24). If bile acid malabsorption were
120100SERUM80-
BILE ACIDS 60.,um/L 40i
20-
,11
T
; I
s6o
~obo
9
I
1so6
BILE ACID SECRETION - ,,umoles/L Fig 3. The relationship b e t w e e n s e r u m bile acid concentration and the corresponding bile acid secretion rate. Normal controls are compared to the patient groups with liver disease: fatty liver of morbid obesity, alcoholic cirrhosis, and cholestasis. M e a n (~ SEM)i s g i v e n for each group. S e r u m levels s h o w e d an inverse correlation with bile acid secretion. Digestive Diseases, Vol. 23, No. 5 (May 1978)
SERUM BILE ACIDS
present, only modest or no serum elevation would be expected. The relationship between bile acid secretion from the liver and the corresponding serum concentrations is clearly altered in cholesterol gallstone disease. Here, a low rate of bile acid secretion, the product of a small circulating bile acid pool (9), is associated with normal serum values. When bile acid secretion is increased in these patients after removal of the gallbladder, serum concentrations remain virtually unchanged. This suggests that hepatic uptake in cholesterol gallstone disease is normal, although a very subtle defect in bile acid flux through the liver cannot be completely eliminated. The high normal serum values present during fasting likely results from a continuous secretion of bile acids into the intestine after cholecystectomy. In contrast to the normal serum bile acids in our patients, the elevated values found by Bell et al (11) may indicate the inclusion of gallstone patients, who can occasionally exhibit biochemical and morphological abnormalities (25). Hence, changes in serum levels reflect the ability of the liver to transport bile acids, but unfortunately do not reveal the abnormality in bile acid metabolism which is associated with cholesterol gallstone disease. ACKNOWLEDGMENTS W e w i s h to t h a n k M s R. O ' B r i e n , M s A. T u o h y , a n d M s K. S a m o d a i for t h e i r e x p e r t t e c h n i c a l a n a l y s i s , M s S. S c h u h a r t for typing this m a n u s c r i p t , a n d Drs. S. B a r n e s a n d U. F a r a c k for t h e i r a d v i c e a n d a s s i s t a n c e .
REFERENCES 1. Carey JB Jr: The serum trihydroxy-dihydroxy bile acid ratio in liver and biliary tract disease. J Clin Invest 37:1494-1503, 1958 2. Sherlock S, Walshe V: Blood cholates in normal subjects and in liver disease. Clin Sci 6:223-234, 1948 3. Osborn EC, Wooton IDP, daSilva LC, Sherlock S. Serumbile-acid levels in liver disease. Lancet 2:1049-1053, 1959 4. Editorial: Serum bile acids as a test of liver function. Lancet 2:267, 1975 5. Kaplowitz N, Kok E, Javitt NB: Postprandial serum bile acid for the d e t e c t i o n of h e p a t o b i l i a r y disease. J A M A 225:292-293, 1973 6. Kaye MD, Struthers TE, Tidball JS, DeNiro E, Kern F Jr: Factors affecting plasma clearance of p4C]cholic acid in patients with cirrhosis. Clin Sci Mol Med 45:147-161, 1973 7. Cowen AE, Korman MG, Hofmann AF, Thomas PJ: Plasma disappearance of radioactivity after intravenous injection of
Digestive Diseases, Vol. 23, No. 5 (May 1978)
labeled bile acids in man. Gastroenterology 68:1567-1573, 1975 8. Korman, MG, LaRusso NF, Hoffman NE, Hofmann AF: Development of an intravenous bile acid tolerance test. Plasma disappearance of cholylglycine in health. N Engl J Med 292:1205-1209, 1975 9. Shaffer EA, Small DM: Biliary lipid secretion in cholesterol gallstone disease: The effect of cholecystectomy and obesity. J Clin Invest 59:828-840, 1977 10. Grundy SM, Metzger AL, Adler RD: Mechanisms of lithogenic bile formation in American Indian women with cholesterol gallstones. J Clin Invest 51:3026-3043, 1972 11. Bell GD, Mok HYL, Thwe M, Murphy GM, Henry K, Dowling RH: Liver structure and function in cholelithiasis: Effect of chenodeoxycholic acid. Gut 15:165-172, 1974 12. Grundy SM, Metzger AL: A physiologic method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology 62:1200-1217, 1972 13. Gordon ER, Shaffer EA, Sass-Kortsak A: Bilimbin secretion and conjugation in the Crigler-Najjar syndrome type II. Gastroenterology 70:761-765, 1976 14. Go VLW, Hofmann AF, Summerskill WHJ: Pancreozymin bioassay in man based on pancreatic enzyme secretion: Potency of specific amino acids and other digestive products. J Clin Invest 49:1558-1564, 1970 15. Fordtran JS: Marker perfusion techniques for measuring intestinal absorption in man. Gastroenterology 51:1089-1093, 1966 16. Seligson D, Marino J, Dodson E: Determination of sulfobromophthalein in serum. Clin Chem 3:638-645, 1957 17. Murphy GM, Billing BH, Baron DN: A fluorimetric and enzymatic method for the estimation of serum total bile acids. J Clin Pathol 123:594-598, 1970 18. Barnes S, Gallo GA, Trash DB, Morris JS: Diagnostic value of serum bile acid estimations in liver disease. J Clin Pathol 28:506-509, 1975 19. Small DM, Dowling RH, Redinger RN: The enterohepatic circulation of bile salts. Arch Intern Med 130:552-573, 1972 20. LaRusso NE, Korman MG, Hofmann NE, Hofmann AF: Dynamics of the enterohepatic circulation of bile acids: Postprandial serum concentrations of conjugates of cholic acid in health, cholecystectomized patients, and patients with bile acid malabsorption. N Engl J Med 291:689-692, 1974 21. Northfield TC, Hofmann AF: Biliary lipid output during three meals and an overnight fast. I Relationship to bile acid pool size and cholesterol saturation of bile in gallstone and control subjects. Gut 16:1-17, 1975 22. Mok HYI, yon Bergmann K, Grundy SM: Effects of intermittent versus continuous feeding on biliary lipid secretion in man. Gastroenterology 71:921, 1976 (abstract) 23. Milstein H J, Bloomer JR, Klatskin G: Serum bile acids in alcoholic liver disease. Am J Dig Dis 21:281-285, 1976 24. Shaffer EA, Beaudoin M, Small DM, O'Brien J, Williams L: Relationship between gallbladder function and the enterohepatic circulation of bile salts during fasting. Gastroenterology 66:775, 1974 25. Fromm H, Holz-Slomczyk M, Zobl H, Schmidt E, Schmidt FW: Studies of liver function and structure in patients with gallstones before and during treatment with chenodeoxycholic acid. Acta Hepato-Gastroenterol 22:359-369, 1975
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