Chemistry and Physics of Lipids, 57 (1991) 97--101 Elsevier Scientific Publishers Ireland Ltd.
97
Short Communication
Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes. II E. D e F a b i a n i , M. C r e s t a n i , L. F a s o l i a n d E. Bosisio Institute of Pharmacological Sciences, Faculty of Pharmacy, University of Milan, via Balzaretti, 9-20133 Milano (Italy) (Received October 17th, 1990; revision received December 5th, 1990; accepted December 6th, 1990)
The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7a-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3a,7Bdibydroxy-20,22-methylen-5B-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 ~M. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7a-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition oftbe analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcyproUDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3a,7B-dibydroxy-22,23-methylen-5B-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase.
Keywords: cholesterol 7¢-hydroxylase; HMGCoA reductase; bile acid analogues; liver microsomes.
Introduction
The natural bile acids are characterized by different physicochemical and biological properties. Several studies to correlate chemical structure, physicochemical and biological properties have been carded out recently. Bile acid structure and conjugation affected bile flow and biliary lipid secretion [1]. Hydrophilicity, critical micellar concentration and conjugation state were shown to affect bile acid hepatic uptake and biliary secretion [2,3]. Bacterial metabolism (e.g. 7-dehydroxylation) [4] and the detergency and dissolution capacity of cholesterol gallstones [5] were also influenced by the chemical structure of bile acids. Recently a correlation was found between hydrophobicity and the capacity of bile acids to inCorrespondenceto: Pro£ Enrica Bosisio, Istituto di Scienze Farmacologiche, Via Balzaretti 9, 20133 Milano, Italy.
hibit the activity of cholesterol 7,-,-hydroxylase in rats treated with different natural bile acids [6]. In a previous study published in this journal [7] we have shown that, in rat liver microsomes, cholesterol 7ot-hydroxylase was differently affected by bile acids depending on the number of hydroxyl groups on the steroid nucleus and the modification of the side-chain structure. It was also reported that, unlike cholesterol 7ot-hydroxylase, HMG-CoA reductase was not inhibited by the tested bile acids [7]. The screening of bile acid analogues to test the effect of the modification of chemical structure on liver cholesterol metabolizing enzymes was further pursued and this report describes the results obtained with 23-methyl-UDCA (3a, 7/~diydroxy-23-methyl-5B-cholan-24-oic acid) and norcypro-UDCA (3ot,7/~-dihydroxy-20,22 methylen-5B-cholan-23-oic acid) on the activity of cholesterol 7ot-hydroxylase. Since the synthesis of
0009-3084/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
98 these bile acids gives rise to a mixture of two isomers [8], the compounds were assayed both as isomeric mixtures and as single isomers.
Bile acids were added as sodium salts at the beginning of the reaction at concentrations ranging from 25 to 200/~M.
Experimental procedures
Stastical analysis Linear regression lines for equation y = a ( ± S.D.) log x + b (q- S.D.) were calculated by least squares method. Regression lines were compared according to the Finney test [12]. Other statistical analyses were performed using one-way analysis of variance and Dunnet test.
Chemicals Bile acids UDCA, 23-MUDCA (23R, S) [8], 23RMUDCA and 23S-MUDCA, norcypro-UDCA (A + B), norcypro-UDCA isomer A and isomer B were kindly supplied by Prof. R. Pelliceiari. 23-MUDCA contained 66% of isomer 23R and 34% of isomer 23S, as determined by GC analysis. Chemical synthesis of norcypro-UDCA will be the subject of a forthcoming paper, which will be submitted for publication elsewhere. The percent composition and the attribution of cis and trans configuration of the two isomers (reported as A and B) of norcyproUDCA are still to be assessed. All other chemicals and solvents, from Merck (Darmstadt, F.R.G.), were of analytical grade. Methods Measurement of cholesterol 7ot-hydroxylase and HMG-CoA reductase Conditions for microsome isolation and enzyme assay were as described in the previous paper [7]. Briefly, male Sprague--Dawley rats (14(b--150 g body weight) were housed under normal light (from 0700 to 1900 h) and were allowed free access to chow diet and water. Animals were killed at 0900 h and livers rapidly removed and placed in ice cold buffer. Final microsomal pellets obtained by ultracentrifugation were suspended in appropriate buffers for cholesterol 7ot-hydroxylase and HMGCoA reductase assays [7]. All procedures were carded out at 4°C and microsomes were kept frozen at -80°C, until use. Protein content was determined according to Bradford [9], using bovine serum albumin as standard. HMG-CoA reductase activity was measured after complete dephosphorylation with a phosphorylase phosphatase partially purified from rat liver [10]. 7ot-Hydroxycholesterol and mevalonic acid formed during the incubation were analyzed by selected ion monitoring [11].
Results and Discussion There is general agreement on the vivo down regulation of cholesterol 7ot-hydroxylase by bile acids returning to the liver through the enterohepatic circulation [4,13], since complete or partial interruption of the enterohepatic circulation was associated with up-regulation of cholesterol 7othydroxylase [14], the rate-limiting enzyme of the overall process of bile acid synthesis. More recent studies suggested that bile acid synthesis could be modulated by the degree of hydrophobicity of bile acids [15] and found a correlation between biliary bile salt hydrophobicity index and the activity of the two enzymes regulating cholesterol and bile acid synthesis, HMG-CoAreductase and cholesterol 7a-hydroxylase [6]. While all these observations were consistent with the concept of negative bile acid feedback, the molecular basis for the regulation of cholesterol 7othydroxylase activity by bile salts remains a debated question. Very recently it was shown that administration of bile acids to rats induced the decrease of levels of mRNA for cholesterol 7othydroxylase [16]. On the other hand, a number of findings in in vitro assay systems failed to account for the occurrence of bile acid feedback. In particular, several groups of investigators were unable to demonstrate inhibition of cholesterol 7othydroxylase activity either in microsomes or in hepatocytes [17--20]. In contrast with these in vitro studies, we found that cholesterol 7a-hydroxylase could be inhibited in rat liver microsomes by the addition of dihydroxy and not by trihydroxy bile acids and that the inhibition was concentration-dependent [7]. Bile acids assayed in the previous study [7] were either natural
100 TABLE II Effect of bile acids on HMG-CoA reductase activity in rat liver microsomes. Bile acids (200 t~M) were added to the incubation mixture just prior to the initiation of the incubation. Activity represents the total form of the enzyme (i.d. after complete dephosphorylation). Microsomal suspension (I 50--200 ~g protein) was incubated in the presence of NADP 2.7 raM, glucose 6-phosphate 25 mM, glucose-6-phosphate dehydrogenase 2 units/ml, 150 ~tM HMGCoA for 30 rain. Bile acids
HMG-CoA reductase activity (pmol/min per mg prot) (mean 4. S.D., n =4)
None UDCA 23R, S-MUDCA 23R-MUDCA 23S-MUDCA Norcypro-UDCA (A + B) Norcypro-UDCA A Norcypro-UDCA B
679 637 611 643 623 668 659 626
± 4. 4. 4. 4. 4. 4. 4.
67 82 55 60 47 20 46 34
When assayed against HMG-CoA-reductase activity, none of the bile acids influenced this enzyme at 200 ~M concentration (Table II). The data presently reported further support the hypothesis that bile acids can modulate cholesterol 7,-hydroxylase when directly added "in vitro". In the light of present and previous reports a dual action of bile acids on their own synthesis might be postulated: either at the transcriptional level [16] by a still unknown mechanism, or by inhibition at enzyme level. Regarding inhibition of the enzyme, several possible mechanisms could be postulated. According to Heuman et al. [15], changes in lipid composition and physicochemical properties of microsomal membranes induced by hydrophobic bile acids could be one possibility. Alternatively, availability of newly synthesized cholesterol may be lowered, due to the inhibition of HMGCoA reductase [22]. A third possibility is the direct effect of bile acids on the enzyme. Our data are not totally consistent with the first hypothesis and the relationship between downregulation of 7o~-hydroxylase and hydrophobicity of bile acids [6].
The order of hydrophobicity of bile acids studied in the present reports is 23S-MUDCA --- UDCA >'23R-MUDCA > norcypro-UDCA ([2], and personal communication by A. Roda). According to the suggested hypothesis, 23S-MUDCA and UDCA which are comparably hydrophobic, should share the same inhibitory activity, which did not happen. Conversely the C24 homologue cyproUDCA (22S, 23S isomer), which is the least hydrophobic, is the most potent among all the tested bile acids [7]. Thus other properties and not just hydrophobicity may be claimed for contributing to the effect. Cholesterol 7o~-hydroxylase has been purified and sequenced only very recently [16,24,25] and little is known about the catalytic site and other conformational and chemico-physical characteristics of the protein, except for an overall hydrophobic nature [16]. When further data on the enzyme protein and on its conformation in relation to the membrane environment become available, a clarification of bile acid structure and enzyme interaction will be feasible. As concerns the second possibility (reduced availability of cholesterol due to inhibition of HMGCoA-reductase), our data cast some doubts on this mechanism since none of the bile acids so far tested inhibited HMGCoA-reductase, when added in vitro. In conclusion, at this stage of knowledge it is difficult to draw some structure-activity relationship and the mechanism by which natural and structurally modified bile acids inhibit cholesterol 7othydroxylase "in vitro" still remains unclear. Further investigations with purified cholesterol 7c~hydroxylase will help in elucidating this stimulating question.
Acknowledgments The authors are indebted to Prof. R. Pellicciari and B. Natalini for the supply of bile acid analogues used in this study.
References 1
D. Gurantz and A.F. Hofmann (1984) Am. J. Physiol. 247, G 738--748.
I01 2
3 4 5 6 7
8 9 10 11
12 13
14
A. Roda, B. Grigolo, E, Roda, P. Simoni, R. PeUicciari, B. Natalini, A. Fini and A.M. Morselli Labate (1988) J. Pharm. Sci. 77, 596--605. R. Aldini, A. Roda, P. Simoni, P. Lenzi and E. Roda (1989) Hepatology 10, 840----845. W.H. BachrachandA.F. Hofmann (1982) Digest. Dis. Sci. 27, 737--856. H. Igimi and M.C. Carey (1981) J. Lipid Res. 22, 254---270. D.M. Heuman, P.B. Hylemon and Z.R. Vlahcevic (1989) J. Lipid Res. 30, 1161--1171. M. Crestani, E. De Fabiani, B. Malavasi, M. Cancellieri, G. Galli and E. Bosisio (1989) Chem. Phys. Lipids 51, 119--126. V. Castagnola, G. Frigerio, R. PeUicciari et al. European patent 0 135 782 A2, 1984. M.M. Bradford (1976) Anal. Biochem. 72, 248---254. G. Cighetti. G. Galli and M. Galli Kienle (1983) Eur. J. Biochem. 133, 573---578. S. BeUentani, M. Pecorari, P. Cordoma, P. Marchegiano, F. Manenti, E. Bosisio, E. De Fabiani and G. Galli (1987) J. Lipids Res. 28, 1021--1027. D.J. Finney (1952) in: Statistical Method in Biological Assay, C. Griffm and Co. Limited, London, pp. 99---136. I. Bjorkem (1985) in: H. Danielson and J. Sjovall (Eds.), Sterols and Bile Acids, Elsevier Scientific Publishing Co., Amsterdam, pp. 231--276. B. Myant and K.A. Mitropoulos (1977) J. Lipid Res. 18, 135--157.
15 D.M. Heuman~ Z.R. Vlahcevic, M.L. Bailey and P.B. Hylemon (1988) Hepatoiogy 8, 892--897. 16 D.F. Jelinek, S. Andersson, C.A. Slanghter and D.W. Russell (1990) J. Lipid Res. 24, 8190--8197: 17 S. Shefer, S. I-laiser and E.H. Mosbach (1968) ,I. Lipid Res. 9, 328--333. 18 R.A. Davis, W.E. Highsmith, J. Malone-McNeal, J. Archambanld-Scheanayder and J.-C.W. Kuan (1983) J. Biol. Chem. 258, 4079----4082. 19 ICM. Botham, M.E. Lawson, G.J. Becket, I.W. Percy-Robb and G.S. Boyd (1981) Biochim. Biophys. Acta 666, 238--245. 20 W.M. Kubaska, E.C. Gurley, P.B. Hylemon, P.S. Guzelian" and Z,R Vlahcevic (1985) Biol. Chem. 200, 13459---13463. 21 M.A. Schwartz and S. Margolis (1983) J. Lipid Res. 24, 28--33. 22 W.M. Pandak, D.M. Heuman, P.B. Hylemon and Z.R. Vlahcevic (1990) J. Lipid Res. 31, 79--90. 23 A. Roda, B. Grigolo, R. Aldini, P. Simoni, R. Pellicciari, B. Natalini and R. Balducci (1987) J. Lipid Res. 28, 1384--1397. 24 Y.C. Li, D.P. Wangand J.Y.L. Chiang(1990) J. Biol. Chem. 265, 12012--12019. 25 M. Noshiro, M. Nishimoto and K. Okuda (1990) J. Biol. Chem. 265, 10036--10041.
97
Short Communication
Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes. II E. D e F a b i a n i , M. C r e s t a n i , L. F a s o l i a n d E. Bosisio Institute of Pharmacological Sciences, Faculty of Pharmacy, University of Milan, via Balzaretti, 9-20133 Milano (Italy) (Received October 17th, 1990; revision received December 5th, 1990; accepted December 6th, 1990)
The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7a-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3a,7Bdibydroxy-20,22-methylen-5B-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 ~M. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7a-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition oftbe analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcyproUDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3a,7B-dibydroxy-22,23-methylen-5B-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase.
Keywords: cholesterol 7¢-hydroxylase; HMGCoA reductase; bile acid analogues; liver microsomes.
Introduction
The natural bile acids are characterized by different physicochemical and biological properties. Several studies to correlate chemical structure, physicochemical and biological properties have been carded out recently. Bile acid structure and conjugation affected bile flow and biliary lipid secretion [1]. Hydrophilicity, critical micellar concentration and conjugation state were shown to affect bile acid hepatic uptake and biliary secretion [2,3]. Bacterial metabolism (e.g. 7-dehydroxylation) [4] and the detergency and dissolution capacity of cholesterol gallstones [5] were also influenced by the chemical structure of bile acids. Recently a correlation was found between hydrophobicity and the capacity of bile acids to inCorrespondenceto: Pro£ Enrica Bosisio, Istituto di Scienze Farmacologiche, Via Balzaretti 9, 20133 Milano, Italy.
hibit the activity of cholesterol 7,-,-hydroxylase in rats treated with different natural bile acids [6]. In a previous study published in this journal [7] we have shown that, in rat liver microsomes, cholesterol 7ot-hydroxylase was differently affected by bile acids depending on the number of hydroxyl groups on the steroid nucleus and the modification of the side-chain structure. It was also reported that, unlike cholesterol 7ot-hydroxylase, HMG-CoA reductase was not inhibited by the tested bile acids [7]. The screening of bile acid analogues to test the effect of the modification of chemical structure on liver cholesterol metabolizing enzymes was further pursued and this report describes the results obtained with 23-methyl-UDCA (3a, 7/~diydroxy-23-methyl-5B-cholan-24-oic acid) and norcypro-UDCA (3ot,7/~-dihydroxy-20,22 methylen-5B-cholan-23-oic acid) on the activity of cholesterol 7ot-hydroxylase. Since the synthesis of
0009-3084/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
98 these bile acids gives rise to a mixture of two isomers [8], the compounds were assayed both as isomeric mixtures and as single isomers.
Bile acids were added as sodium salts at the beginning of the reaction at concentrations ranging from 25 to 200/~M.
Experimental procedures
Stastical analysis Linear regression lines for equation y = a ( ± S.D.) log x + b (q- S.D.) were calculated by least squares method. Regression lines were compared according to the Finney test [12]. Other statistical analyses were performed using one-way analysis of variance and Dunnet test.
Chemicals Bile acids UDCA, 23-MUDCA (23R, S) [8], 23RMUDCA and 23S-MUDCA, norcypro-UDCA (A + B), norcypro-UDCA isomer A and isomer B were kindly supplied by Prof. R. Pelliceiari. 23-MUDCA contained 66% of isomer 23R and 34% of isomer 23S, as determined by GC analysis. Chemical synthesis of norcypro-UDCA will be the subject of a forthcoming paper, which will be submitted for publication elsewhere. The percent composition and the attribution of cis and trans configuration of the two isomers (reported as A and B) of norcyproUDCA are still to be assessed. All other chemicals and solvents, from Merck (Darmstadt, F.R.G.), were of analytical grade. Methods Measurement of cholesterol 7ot-hydroxylase and HMG-CoA reductase Conditions for microsome isolation and enzyme assay were as described in the previous paper [7]. Briefly, male Sprague--Dawley rats (14(b--150 g body weight) were housed under normal light (from 0700 to 1900 h) and were allowed free access to chow diet and water. Animals were killed at 0900 h and livers rapidly removed and placed in ice cold buffer. Final microsomal pellets obtained by ultracentrifugation were suspended in appropriate buffers for cholesterol 7ot-hydroxylase and HMGCoA reductase assays [7]. All procedures were carded out at 4°C and microsomes were kept frozen at -80°C, until use. Protein content was determined according to Bradford [9], using bovine serum albumin as standard. HMG-CoA reductase activity was measured after complete dephosphorylation with a phosphorylase phosphatase partially purified from rat liver [10]. 7ot-Hydroxycholesterol and mevalonic acid formed during the incubation were analyzed by selected ion monitoring [11].
Results and Discussion There is general agreement on the vivo down regulation of cholesterol 7ot-hydroxylase by bile acids returning to the liver through the enterohepatic circulation [4,13], since complete or partial interruption of the enterohepatic circulation was associated with up-regulation of cholesterol 7othydroxylase [14], the rate-limiting enzyme of the overall process of bile acid synthesis. More recent studies suggested that bile acid synthesis could be modulated by the degree of hydrophobicity of bile acids [15] and found a correlation between biliary bile salt hydrophobicity index and the activity of the two enzymes regulating cholesterol and bile acid synthesis, HMG-CoAreductase and cholesterol 7a-hydroxylase [6]. While all these observations were consistent with the concept of negative bile acid feedback, the molecular basis for the regulation of cholesterol 7othydroxylase activity by bile salts remains a debated question. Very recently it was shown that administration of bile acids to rats induced the decrease of levels of mRNA for cholesterol 7othydroxylase [16]. On the other hand, a number of findings in in vitro assay systems failed to account for the occurrence of bile acid feedback. In particular, several groups of investigators were unable to demonstrate inhibition of cholesterol 7othydroxylase activity either in microsomes or in hepatocytes [17--20]. In contrast with these in vitro studies, we found that cholesterol 7a-hydroxylase could be inhibited in rat liver microsomes by the addition of dihydroxy and not by trihydroxy bile acids and that the inhibition was concentration-dependent [7]. Bile acids assayed in the previous study [7] were either natural
100 TABLE II Effect of bile acids on HMG-CoA reductase activity in rat liver microsomes. Bile acids (200 t~M) were added to the incubation mixture just prior to the initiation of the incubation. Activity represents the total form of the enzyme (i.d. after complete dephosphorylation). Microsomal suspension (I 50--200 ~g protein) was incubated in the presence of NADP 2.7 raM, glucose 6-phosphate 25 mM, glucose-6-phosphate dehydrogenase 2 units/ml, 150 ~tM HMGCoA for 30 rain. Bile acids
HMG-CoA reductase activity (pmol/min per mg prot) (mean 4. S.D., n =4)
None UDCA 23R, S-MUDCA 23R-MUDCA 23S-MUDCA Norcypro-UDCA (A + B) Norcypro-UDCA A Norcypro-UDCA B
679 637 611 643 623 668 659 626
± 4. 4. 4. 4. 4. 4. 4.
67 82 55 60 47 20 46 34
When assayed against HMG-CoA-reductase activity, none of the bile acids influenced this enzyme at 200 ~M concentration (Table II). The data presently reported further support the hypothesis that bile acids can modulate cholesterol 7,-hydroxylase when directly added "in vitro". In the light of present and previous reports a dual action of bile acids on their own synthesis might be postulated: either at the transcriptional level [16] by a still unknown mechanism, or by inhibition at enzyme level. Regarding inhibition of the enzyme, several possible mechanisms could be postulated. According to Heuman et al. [15], changes in lipid composition and physicochemical properties of microsomal membranes induced by hydrophobic bile acids could be one possibility. Alternatively, availability of newly synthesized cholesterol may be lowered, due to the inhibition of HMGCoA reductase [22]. A third possibility is the direct effect of bile acids on the enzyme. Our data are not totally consistent with the first hypothesis and the relationship between downregulation of 7o~-hydroxylase and hydrophobicity of bile acids [6].
The order of hydrophobicity of bile acids studied in the present reports is 23S-MUDCA --- UDCA >'23R-MUDCA > norcypro-UDCA ([2], and personal communication by A. Roda). According to the suggested hypothesis, 23S-MUDCA and UDCA which are comparably hydrophobic, should share the same inhibitory activity, which did not happen. Conversely the C24 homologue cyproUDCA (22S, 23S isomer), which is the least hydrophobic, is the most potent among all the tested bile acids [7]. Thus other properties and not just hydrophobicity may be claimed for contributing to the effect. Cholesterol 7o~-hydroxylase has been purified and sequenced only very recently [16,24,25] and little is known about the catalytic site and other conformational and chemico-physical characteristics of the protein, except for an overall hydrophobic nature [16]. When further data on the enzyme protein and on its conformation in relation to the membrane environment become available, a clarification of bile acid structure and enzyme interaction will be feasible. As concerns the second possibility (reduced availability of cholesterol due to inhibition of HMGCoA-reductase), our data cast some doubts on this mechanism since none of the bile acids so far tested inhibited HMGCoA-reductase, when added in vitro. In conclusion, at this stage of knowledge it is difficult to draw some structure-activity relationship and the mechanism by which natural and structurally modified bile acids inhibit cholesterol 7othydroxylase "in vitro" still remains unclear. Further investigations with purified cholesterol 7c~hydroxylase will help in elucidating this stimulating question.
Acknowledgments The authors are indebted to Prof. R. Pellicciari and B. Natalini for the supply of bile acid analogues used in this study.
References 1
D. Gurantz and A.F. Hofmann (1984) Am. J. Physiol. 247, G 738--748.
I01 2
3 4 5 6 7
8 9 10 11
12 13
14
A. Roda, B. Grigolo, E, Roda, P. Simoni, R. PeUicciari, B. Natalini, A. Fini and A.M. Morselli Labate (1988) J. Pharm. Sci. 77, 596--605. R. Aldini, A. Roda, P. Simoni, P. Lenzi and E. Roda (1989) Hepatology 10, 840----845. W.H. BachrachandA.F. Hofmann (1982) Digest. Dis. Sci. 27, 737--856. H. Igimi and M.C. Carey (1981) J. Lipid Res. 22, 254---270. D.M. Heuman, P.B. Hylemon and Z.R. Vlahcevic (1989) J. Lipid Res. 30, 1161--1171. M. Crestani, E. De Fabiani, B. Malavasi, M. Cancellieri, G. Galli and E. Bosisio (1989) Chem. Phys. Lipids 51, 119--126. V. Castagnola, G. Frigerio, R. PeUicciari et al. European patent 0 135 782 A2, 1984. M.M. Bradford (1976) Anal. Biochem. 72, 248---254. G. Cighetti. G. Galli and M. Galli Kienle (1983) Eur. J. Biochem. 133, 573---578. S. BeUentani, M. Pecorari, P. Cordoma, P. Marchegiano, F. Manenti, E. Bosisio, E. De Fabiani and G. Galli (1987) J. Lipids Res. 28, 1021--1027. D.J. Finney (1952) in: Statistical Method in Biological Assay, C. Griffm and Co. Limited, London, pp. 99---136. I. Bjorkem (1985) in: H. Danielson and J. Sjovall (Eds.), Sterols and Bile Acids, Elsevier Scientific Publishing Co., Amsterdam, pp. 231--276. B. Myant and K.A. Mitropoulos (1977) J. Lipid Res. 18, 135--157.
15 D.M. Heuman~ Z.R. Vlahcevic, M.L. Bailey and P.B. Hylemon (1988) Hepatoiogy 8, 892--897. 16 D.F. Jelinek, S. Andersson, C.A. Slanghter and D.W. Russell (1990) J. Lipid Res. 24, 8190--8197: 17 S. Shefer, S. I-laiser and E.H. Mosbach (1968) ,I. Lipid Res. 9, 328--333. 18 R.A. Davis, W.E. Highsmith, J. Malone-McNeal, J. Archambanld-Scheanayder and J.-C.W. Kuan (1983) J. Biol. Chem. 258, 4079----4082. 19 ICM. Botham, M.E. Lawson, G.J. Becket, I.W. Percy-Robb and G.S. Boyd (1981) Biochim. Biophys. Acta 666, 238--245. 20 W.M. Kubaska, E.C. Gurley, P.B. Hylemon, P.S. Guzelian" and Z,R Vlahcevic (1985) Biol. Chem. 200, 13459---13463. 21 M.A. Schwartz and S. Margolis (1983) J. Lipid Res. 24, 28--33. 22 W.M. Pandak, D.M. Heuman, P.B. Hylemon and Z.R. Vlahcevic (1990) J. Lipid Res. 31, 79--90. 23 A. Roda, B. Grigolo, R. Aldini, P. Simoni, R. Pellicciari, B. Natalini and R. Balducci (1987) J. Lipid Res. 28, 1384--1397. 24 Y.C. Li, D.P. Wangand J.Y.L. Chiang(1990) J. Biol. Chem. 265, 12012--12019. 25 M. Noshiro, M. Nishimoto and K. Okuda (1990) J. Biol. Chem. 265, 10036--10041.
