Cholestyramine Treatment Reduces Postprandial but not Fasting Serum Bile Acid Levels in Humans

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1 GASTROENTEROLOGY 1982;83: Cholestyramine Treatment Reduces Postprandial but not Fasting Serum Bile Acid Levels in Humans BO ANGELIN, INGEMAR BJORKHEM, KURT EINARSSON, and STAFFAN EWERTH The Departments of Medicine, Clinical Chemistry, and Surgery, Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden Fasting serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid were determined in healthy subjects and in patients with familial hypercholesterolemia before and during treatment with cholestyramine. The bile acids were analyzed by a specific isotope-dilution technique by using gas chromatography-mass spectrometry. Cholestyramine treatment did not change the fasting concentration of total bile acids, but the contribution of cholic acid was increased; those of chenodeoxycholic acid and deoxycholic acid were decreased. No decrease of fasting bile-acid concentrations in portal venous serum was seen in 2 cholestyramine-treated gallstone patients. The postprandial total bile-acid concentration was about 40% lower during cholestyramine treatment in healthy subjects, reflecting a reduced postprandial inflow of bile acids to the liver. This degree of interruption of the postprandial enterohepatic circulation may be sufficient to produce a n ear maximal bile-acid biosynthesis rate and to promote lowering of plasma cholesterol also in the fasting state. It is concluded that the postprandial bile-acid inflow to the liver may be more important as a regulator of bile-acid biosynthesis than is the fasting level of bile acids. Received July 29, Accepted June 4, Address requests for reprints to: Bo Angelin, M.D., Department of Medicine, Huddinge University Hospital, S Huddinge, Sweden. This study was supported by grants from the Karolinska Institutet, the Loo and Hans Osterman Foundation, and from the Swedish Medical Research Council (03X and 03X-3141). Dr. Angelin is the recipient of a fellowship from the Ernst Klenk Foundation. The authors a re grateful to Ms. Helene Saranius and Ms. Gunvor Alvelius for their expert technical assistance, and to Ms. Lena Ericsson for her skillful preparation of the manuscript by the American Gastroenterological Association /82/ $02.50 In humans, the two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), are formed in the liver from cholesterol. Together with the secondary bile acid, deoxycholic acid (DCA), they are secreted as taurine and glycine conjugates in the bile. The bile acids are partly stored in the gallbladder and released into the intestine in response to a meal. After promoting fat absorption, bile acids are efficiently reabsorbed and recirculated to the liver via the portal vein. Due to the highly efficient hepatic uptake, only small amounts of bile acids are generally detected in the peripheral circulation (1,2). Because of a constant fractional uptake over a wide range of bile acid concentrations, the peripheral venous concentration of a bile acid reflects its portal venous concentration (2). There is considerable variation in the inflow of bile acids to the liver, with three- to fivefold increases of serum bile-acid concentration in peripheral circulation after a meal (3,4). The amount of bile acids returning to the liver is assumed to regulate the conversion of cholesterol to bile acids (negative feedback) (5). Animal studies suggest that there is a diurnal variation in the biosynthesis of bile acids, with a higher synthetic activity during the feeding period (5). No information is available about humans, however, as to whether the fasting or the postprandial bile-acid inflow is the major regulator of bile-acid production rate. In the present work, we have used the experimental model of cholestyramine feeding to study how bile-acid return may regulate bile-acid synthesis. Oral administration of this bile-acid-binding resin is accompanied by an increased fecal excretion of bile acids (6,7). The increased loss of bile acids is counterbalanced by an enhanced conversion of cholesterol to bile acids in the liver (8-10), resulting in a decrease of fasting plasma cholesterol levels (11,12). We specifically tested the hypothesis that cholesty-

2 1098 ANGELIN ET AL. GASTROENTEROLOGY Vol. 83, No. 5 ramine exerts its effects on bile-acid and cholesterol metabolism by reducing the fasting inflow of bile acids to the liver. This was achieved by determining serum bile-acid levels both in the fasting state and in response to a standardized meal before and during therapy with cholestyramine. The results do not favor the concept of an important role for the fasting inflow of bile acids, but instead point to the postprandial bile-acid return as a major regulator of bileacid and cholesterol metabolism. Material and Methods Subjects and Experimental Procedures Three groups of subjects were studied under three different experimental protocols. (a) Ten patients with heterozygous familial hypercholesterolemia were studied before and during treatment with cholestyramine (Questran, Mead Johnson Pharmaceutical, Evansville, Ind.). The drug was administered at a dosage of 8 g h.ld. for 8 wk. Fasting plasma lipid and serum bile-acid levels were determined after a 12-h fast. (b) Two patients with uncomplicated cholesterol gallstones were studied during cholecystectomy after 3 wk of treatment with cholestyramine (8 g b.ld.). Portal venous blood was obtained and serum bile acids determined (ef Reference 2). (c) Eight healthy volunteers were investigated both fasting and postprandially before and during treatment with cholestyramine at a similar dose. Two blood samples were drawn from a needle inserted into an antecubital vein on the morning after a 12-h fast. The subjects were then given a standardized meal consisting of 2 dl milk, 2 cheese sandwiches, and 2 dl coffee. The energy content of this meal is 1750 kj (33% carbohydrate, 44% fat, and 23% protein). Venous blood samples were drciwn at 15-min intervals for 210 min. During the study preceded by 3 wk of treatment with cholestyramine, the subjects ingested 8 g of cholestyramine immediately preceding the meal. The ethical aspects of the present study were approved by the Ethical Committee of Karolinska Institutet on March 3, Methods Venous blood samples were allowed to clot at room temperature. Serum was obtained by centrifugation, and it was frozen at -20 C for subsequent analysis. To 0.5 ml of serum, 1 I-Lg of [2,2,3,4,4-2 Hslcholic acid, 0.5 I-Lg of [2,2,3,4,4- z Hslchenodeoxycholic acid, and 2.5 I-Lg of [11,11,12- z H 3 1deoxycholic acid were added at the same time. The serum together with the internal standards was hydrolyzed with 1 M KOH at 110 C for 12 h. The alkaline solutiqn was extracted three times with diethyl ether in order to remove most of the neutral steroids. The bile acids were then extracted from the acidified water phase with ethyl ether, methylated with diazomethane, and converted into trimethyl-silyl ether derivatives. The derivates were analyzed by gas chromatography-mass spectrometry using an LKB 9000 instrument equipped with multiple ion detector (MID) unit (LKB Instruments, Rockville, Md.). A 1.5% SE-30 column was used and the operative temperature was C. Our mass-fragmentographic technique (13) for determination of the individual bile acids, CA, CDCA, and DCA, in serum has been modified, using more specific ions for each bile-acid derivative. As a consequence, however, each bile acid must be determined separately with the instrument used here (LKB 9000). Thus, CA was analyzed with two of the channels focused on mle 623 and mle 628, corresponding to the M-15 peak in the mass spectrum of trimethyl-silyl derivative of unlabeled and [ZH1CA, respectively. Chenodeoxycholic acid was analyzed with two of the channels focused on mle 370 and 373, corresponding to the M-2 x 90 peak in the mass spectrum of trimethylshyl derivative of unlabeled and [ZH1CDCA, respectively. Deoxycholic acid was analyzed with two of the channels focused on mle 255 and 258, corresponding to the base peak in the mass spectrum of trimethyl-silyl derivative of unlabeled and [ZHIDCA, respectively. The concentrations of the individual bile acids were thus calculated from the ratios CA/[2,2, z HslCA (ratio of tracing at mle 623 to tracing at mle 628), CDCA/[2,2,3,4.4- z HslCDCA (ratio of tracing at mle 370 to tracing at mle 373), and DCAI [11,11,12- z H 3 1DCA (ratio of tracing at mle 255 to tracing at mle 258). Standard curves were used for each bile acid. As calculated from duplicate samples, the relative standard deviation of the method was 2%-3%. Plasma lipid and lipoprotein concentrations were determined as described previously (14). Data are presented as means ± standard error of the mean (SEM). Significances of differences were evaluated by Student's paired t-test (15). Results Cholestyramine treatment reduced fasting plasma total cholesterol and low-density-lipoprotein (LDL) cholesterol by about 30% in patients with familial hypercholesterolemia. Fasting serum total bile acid concentration was not changed significantly by therapy, but the concentration of CA was increased and that of DCA was reciprocally decreased (Table 1). This indicated that the portal venous inflow of bile acids was not reduced by cholestyramine treatment in the fasting state. To directly assess this, we determined the fasting concentrations of CA, CDCA, and DCA in portal venous blood from 2 cholestyramine-treated gallstone patients (Table 2). Also in this case, the relative amount of CA appeared to be increased and that of DCA reduced, whereas the total bile-acid concentration was within the normal range observed in our previously published study (2). Thus, cholestyramine treatment clearly reduced fasting total- and LDLcholesterol levels without reducing the fasting portal venous inflow of bile acids. These findings led us to formulate and test the revised hypothesis that cholestyramine exerts its

3 November 1982 CHOLESTYRAMINE TREATMENT AND SERUM BILE ACIDS 1099 Table 1. Effects of Cholestyramine Treatment G on Fasting Plasma Lipid and Serum Bile Acid Levels in Ten Patients With Heterozygous Familial Hypercholesterolemia Basal b During treatment b Cholesterol (mmolll) 9.3 ± ± 0.3" Triglycerides (mmolll) 1.6 ± ± 0.2 LDL cholesterol (mmolll) 7.0 ± ± O.4 c Cholic acid (J.LmollL) 0.37 ± ± 0.21 d Chenodeoxycholic acid (J.LmollL) 0.87 ± ± 0.14 Deoxycholic acid (J.LmollL) 0.94 ± ± 0.07 d Total bile acids(j.lmolll) 2.18 ± ± 0.34 a Dosage: 16 g daily for 8 wk. b Mean ± SEM. C Significantly different from basal period, p < d P < major effect on bile-acid enterohepatic circulation in the postprandial state. In 8 healthy subjects, cholestyramine reduced total plasma cholesterol by 20%, whereas again it did not change the fasting concentration of total bile acids (Table 3). As previously observed in hypercholesterolemic patients, the concentration of CA was increased and that of DCA decreased. With cholestyramine therapy, the postprandial increase in serum bile acids was both qualitatively and quantitatively different (Figure 1). Thus, the normal postprandial dominance of the dihydroxy bile acids, CDCA and DCA, in peripheral serum was abolished by cholestyramine treatment, and CA was the major postprandial bile acid (Table 3). After meals, the sum of CA, CDCA, and DCA was clearly reduced during cholestyramine therapy (Figure 2, Table 3). This decrease was also confirmed by calculating the mean area under the curves of total bile-acid concentrations, which showed a 40% reduction (p < 0.02). All the previous studies were performed with a dosage regimen used in clinical practice. It could be argued that the lack of effect on fasting bile-acid levels is due to the fact that only minor amounts of Table 3. Effects of Cholestyramine Treatment G on Fasting Plasma Lipid and Fasting and Postprandial Serum Bile Acid Levels in Eight Healthy Subjects Basal b During treatment b Fasting plasma lipids c Cholesterol 5.0 ± ±0.3 D Triglycerides 1.1 ± ± 0.5 Fasting serum bile acids d Cholic acid 0.24 ± ± Chenodeoxycholic acid 0.77 ± ± 0.06 Deoxycholic acid 0.82 ± ± 0.08 g Total bile acids 1.85 ± ± 0.28 Postprandial maximum d Cholic acid 0.79 ± ± 0.29 Chenodeoxycholic acid 2.78 ± ± 0.1O! Deoxycholic acid 1.54 ± ± 0.09! Total bile acids 5.69 ± ± 0.31 g Dosage: 16 g daily for 3 wk. b Mean ± SEM. C In millimoles per liter. d In micromoles per liter. e Significantly different from basal period, p < f P < g P < cholestyramine are probably present in the gut in the fasting state. The results from two additional experiments did not favor this view, however. First, when fasting serum bile acids were determined in 6 healthy subjects treated with 8 g of cholestyramine twice daily for 3 wk, the results were not different if the latter dose was given 5-6 h after the last meal of the preceding day, i.e., 7-8 h before the fasting sample was taken (2.05 ± 0.51 vs ± 0.34 /Lmoll L). Second, when hourly fasting samples were drawn after the ingestion of 8 g of cholestyramine in 6 healthy subjects (treated for 3 wk), there was no significant decrease in the total serum bile-acid concentration over a period of 5 h. Furthermore, the mean total bile-acid concentration during the 5-h fast with cholestyramine in these subjects (1.08 ± 0.19 /LmollL) was not significantly different from that seen during a similar fast without administration of the drug (1.23 ± 0.13 /LmollL). Table 2. Portal Venous Concentration of Individual Bile Acids in Two Normal Gallstone Patients Treated With Cholestyramine for Three Weeks Chenodeoxycholic Deoxycholic Total bile Cholic acid acid acid acids (J.Lmol/L) (J.LmollL) (J.LmollL) (J.LmollL) Patients Untreated patients O (Range) ( ) ( ) ( ) ( ) Data from Reference 2; n = 10.

4 1100 ANGELIN ET AL. GASTROENTEROLOGY Vol. 83, No. 5, A. B. "0 E -=-3 c:: 0 ~ c:: Q) u 5 u., "C 0.!! :c E ::l 4i III o Time Imin.! Time Iminl Figure 1. Postprandial concentrations of cholic acid (e-e), chenodeoxycholic acid (A-A), and deoxycholic acid (.-.) in 1 healthy subject during the control period (A) and during cholestyramine treatment (B). Discussion The present isotope-dilution-mass spectrometric technique, using more specific ions for each bile-acid derivative, is even more accurate than the previously used method (13). Under the conditions employed, no information was obtained with respect to the degree and nature of conjugation or sulfation of the bile acids. In this study, however, these limitations should be of minor importance. Using three different experimental protocols, we did not find any support for the hypothesis that the effects of cholestyramine on bile-acid and cholesterol metabolism are due to a reduced fasting bile-acid inflow to the liver. In all studies cholestyramine did produce qualitative changes, however, in that CA became the dominant serum bile acid. These results are in accordance with previous reports on fasting gallbladder bile composition (10,16,17), and they reflect a changed composition of the bile acid pool (8,10). It could be argued that this change in the composition of bile acids returning to the liver could be an explanation of the reduced feedback inhibition of bile-acid synthesis seen during cholestyramine treatment. However, there is presently no evidence of a difference in inhibitory capacity with regard to bile-acid production between the individual bile acids in humans (18-20). Clear changes in postprandial bile-acid inflow to the liver were brought about by cholestyramine treatment. The postprandial total bile-acid concentration was reduced by about 40%, mainly due to a reduced postprandial rise in CDCA and DCA. Cholestyramine has a greater affinity for dihydroxy than for trihydroxy bile acids in vitro (21,22). Thus, the degree of interruption of the enterohepatic circulation will be higher for CDCA and DCA than for CA. This effect will be further increased by the fact that CDCA and DCA normally are taken up more efficiently than CA in the upper intestine (23) and thus circulate faster through the enterohepatic circulation (24). The present study leads us to postulate that the powerful effects of cholestyramine treatment on cholesterol metabolism in humans are mainly the consequences of postprandial, and not fasting, interruption of bile-acid enterohepatic circulation. It is of interest to note that studies in the rhesus monkey suggest that a 20% diversion of bile flow will result in a maximum stimulation of bile-acid biosynthesis (25). Thus, the 40% reduction of the postprandial o Time Imin.! Figure 2. Means of total postprandial serum bile-acid concentrations (sum of cholic acid, chenodeoxycholic acid, and deoxycholic acid) in 8 healthy subjects during control period (_-_) and during cholestyramine treatment (e-e). Bars indicate SEM. *p < 0.05, **p < 0.01.

5 November 1982 CHOLESTYRAMINE TREATMENT AND SERUM BILE ACIDS 1101 bile-acid inflow that we recorded in the present study-using therapeutic dosages of cholestyramine-may well be sufficient to promote maximal or near-maximal stimulation of bile-acid biosynthesis during the period of the day when basal production rate is believed to be highest (5). Secondary metabolic adaptations, such as induction of hepatic lipoprotein receptors and enhanced LDL degradation (26), probably involve more slowly adjustable mechanisms and may thus persist also in the fasting state. Thus, it may be speculated that relatively transient changes in the transhepatic flux of bile acids postprandially may result in sustained effects on plasma lipoprotein metabolism. References 1. Hofmann AF. The enterohepatic circulation of bile acids in man. Adv Intern Med 1976;21: Ahlberg 1. Angelin B, Bjorkhem I, et al. Individual bile acids in portal venous and systemic blood serum of fasting man. Gastroenterology 1977;73: LaRusso NF, Korman MG, Hoffman NE, et al. 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 1974;291: Angelin B, Bjorkhem 1. Postprandial serum bile acids in healthy man-evidence for differences in absorptive pattern between individual bile acids. Gut 1977;18: Danielsson H, Sjovall J. Bile acid metabolism. Annu Rev Biochem 1975;33: Grundy SM, Ahrens EH, Salen G. Interruption of the enterohepatic circulation of bile acids in man: comparative effects of cholestyramine and ileal exclusion on cholesterol metabolism. J Lab Clin Med 1971;78: Miettinen T. Bile acid metabolism. In: Kritchevsky 0, ed. Hypolipidemic agents. Berlin-Heidelberg-New York: Springer Verlag, 1975; Einarsson K, Hellstrom K, Kallner M. The effect of cholestyramine on the elimination of cholesterol as bile acids in patients with hyperlipoproteinaemia type II and IV. Eur J Clin Invest 1974;4: Garbutt JT, Kenney TJ. Effect of cholestyramine on bile acid metabolism in normal man. J Clin Invest 1972;51: Andersen E. The effect of cholestyramine on bile acid kinetics in healthy controls. Scand J GastroenteroI1979;14: Jones R1. Dobrilovic 1. Lipoprotein alterations with cholestyramine administration. J Lab Clin Med 1970;75: Levy RI, Fredrickson OS, Stone N1. et al. Cholestyramine in type II hyperlipoproteinemia-a double-blind trial. Ann Intern Med 1973;79: Angelin B, Bjorkhem I, Einarsson K. Individual serum bile acid concentrations in normo- and hyperlipoproteinemia as determined by mass fragmentography: relation to bile acid pool size. J Lipid Res 1978;19: Angelin B, Einarsson K, Leijd B. Clofibrate treatment and bile cholesterol saturation: short-term and long-term effects and influence of combination with chenodeoxycholic acid. Eur J Clin Invest 1981;11: Snedecor GW, Cochran WG. Statistical methods. 6th ed. Ames, Iowa: Iowa State University Press, Wood PO, Shioda R, Estrich DL, et al. Effect of cholestyramine on composition of duodenal bile in obese human subjects. Metabolism 1972;21: Angelin B, Einarsson K, Leijd B. Biliary lipid composition during treatment with different hypolipidaemic drugs. Eur J Clin Invest 1979;9: Einarsson K, Hellstrom K, Kallner M. Feedback regulation of bile acid formation in man. Metabolism 1973;22: LaRusso NF, Hoffman NE, Hofmann AF, et al. Effect of primary bile acid ingestion on bile acid metabolism and biliary lipid secretion in gallstone patients. Gastroenterology 1975;68: Kallner M. The effect of chenodeoxycholic acid feeding on bile acid kinetics and fecal neutral steroid excretion in patients with hyperlipoproteinemia types II and IV. J Lab Clin Med 1975;86: Johns WH, Bates TR. Quantification of the binding tendencies of cholestyramine. 1. Effect of structure and added electrolytes on the binding of unconjugated and conjugated bile-salt anions. J Pharm Sci 1969;58: Thale M, and Faergeman O. Binding of bile acids to anionexchanging drugs in vitro. Scand J Gastroenterol 1978; 13: Angelin B, Einarsson K, Hellstrom K. Evidence for the absorption of bile acids in the proximal small intestine of normoand hyperlipidaemic subjects. Gut 1976;17: Einarsson KA, Grundy SM, Hardison WGM. Enterohepatic circulation rates of cholic acid and chenodeoxycholic acid in man. Gut 1979;20: Dowling RH, Mack E, Small OM. Effects of controlled interruption of the enterohepatic circulation of bile salts by biliary diversion and by ileal resection on bile salt secretion, synthesis, and pool size in the rhesus monkey. J Clin Invest 1970; 49: Brown MS, Kovanen PT, Goldstein JL. Regulation of plasma cholesterol by lipoprotein receptors. Science 1981;212: