Bile Acid Synthesis in Cultured Human Hepatocytes: Support for an Alternative Biosynthetic Pathway to Cholic Acid

Transcription

1 Bile Acid Synthesis in Cultured Human Hepatocytes: Support for an Alternative Biosynthetic Pathway to Cholic Acid MAGNUS AXELSON, 1 EWA ELLIS, 2 BIRGITTA MÖRK, 1 KRISTINA GARMARK, 1 ANNA ABRAHAMSSON, 2 INGEMAR BJÖRKHEM, 3 BO-GÖRAN ERICZON, 4 AND CURT EINARSSON 2 The biosynthesis of bile acids by primary cultures of normal human hepatocytes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange chromatography and gas chromatography mass spectrometry (GC/MS). Following incubation for 5 days, 8 oxysterols and8c 27 -orc 24 -bile acids were identified in media and cells. Cholic and chenodeoxycholic acids conjugated with glycine or taurine were by far the major steroids found, accounting for 70% and 24% of the total, respectively, being consistent with bile acid synthesis in human liver. Small amounts of sulfated 3 -hydroxy-5-cholenoic acid and 3,7 dihydroxy-5 -cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7,12 -dihydroxy-3-oxo-4-cholenoic acid and its 5 -reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7 hydroxylation of cholesterol, followed by oxidation to 7 -hydroxy-4-cholesten-3-one and 12 -hydroxylation. Notably, 27-hydroxylation of the product 7,12 -dihydroxy-4- cholesten-3-one and further oxidation and cleavage of the side chain precede A-ring reduction. A-Ring reduction may also occur before side-chain cleavage, but after 27- hydroxylation, yielding 3,7,12 -trihydroxy-5 -cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7,12 dihydroxy-4-cholesten-3-one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates reflect an alternative biosynthetic pathway to CA, which may be quantitatively significant in the cells. (HEPATOLOGY 2000;31: ) Cholic acid (CA) and chenodeoxycholic acid (CDCA) are the major catabolic end products of cholesterol metabolism in the human liver. They may be formed via several different pathways as demonstrated by administration of potential intermediates and by studies of enzyme kinetics in vitro. 1-6 However, the most important biosynthetic pathways to the 2 bile acids in vivo have been difficult to establish mainly as a result of the many enzymes involved with broad substrate specificities. Furthermore, a rate-limiting hydroxylation of cholesterol in the 7 - or 27-position is followed by a rapid conversion into bile acids, resulting in low and usually undetectable concentrations of intermediates in liver tissue. 7 A different approach in studying bile acid synthesis is to use cultured hepatocytes. 8,9 We have previously studied the formation of these compounds in cultured human hepatoblastoma cells (HepG2). These cells were found to release relatively large amounts of bile acid precursors into the incubation medium, and based on this finding, major biosynthetic pathways to CA and CDCA in HepG2 cells could be elucidated. 10 However, we could not exclude that the tumor cells converted cholesterol to bile acids via pathways different from those in normal hepatocytes. More recently, techniques have been developed for preparing primary cultures of normal human hepatocytes. 9 Thus, it is now possible to study bile acid synthesis in normal cells under various experimental conditions. 11,12 Here, we report the results of such a study showing that the behavior of cultured normal human hepatocytes closely resembles that of intact liver cells, although the order of reactions for CA synthesis appeared to be different from that generally believed to exist in humans. Abbreviations: CA, cholic acid (3,7,12 -trihydroxy-5 -cholanoate); CDCA, chenodeoxycholic acid (3,7 -dihydroxy-5 -cholanoate); CsA, cyclosporin A; ODS, octadecylsilane; TEAP-LH-20, triethylaminohydroxypropyl-sephadex LH-20; GLC, gas-liquid chromatography; GC/MS, gas chromatography mass spectrometry; THCA, 3,7,12 -trihydroxy-5 -cholestanoate. From the 1 Department of Clinical Chemistry, Karolinska Hospital and the Departments of 2 Medicine, 3 Clinical Chemistry, and 4 Transplantation Surgery, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden. Received January 5, 2000; accepted March 21, Supported by the Swedish Medical Research Council (grants 03X-7890, 03X-4793, and 03X-3141) and the Karolinska Institutet. Address reprint requests to: Magnus Axelson, M.D., Department of Clinical Chemistry, Karolinska Hospital, S Stockholm, Sweden. magnus.axelson@lab.ks.se; fax: Copyright 2000 by the American Association for the Study of Liver Diseases /00/ $3.00/0 doi: /jhep MATERIALS AND METHODS Steroids, Chemicals, and Column Packing Materials. Reference steroids used for the identification and quantitation of neutral sterols and C 27 - and C 24 -bile acids were those described in previous studies. 10,13,14 Isotopically labeled steroids, added to samples before purification and used for correction of losses, were: [ 2 H 7 ]27- hydroxycholesterol, 13 [2,4 3 H]cholic acid, and [24-14 C]taurocholic acid (from NEN Life Science Products, Inc., Boston, MA); and [ 3 H]glycochenodeoxycholic acid monosulfate (a gift from Dr. G. Hedenborg, Stockholm, Sweden). Cyclosporin A (CsA) was a gift from Sandoz Pharma Ltd. (Basel, Switzerland). Solvents were of analytical reagent grade. Choloylglycine hydrolase (from Clostridium perfringens) and 2-mercaptoethanol were from Sigma Chemical Co. (St. Louis, MO). Titriplex III was from Merck (Darmstadt, Germany). Diazomethane was freshly prepared 1305

2 1306 AXELSON ET AL. HEPATOLOGY June 2000 from Diazald (Sigma-Aldrich, Steinhelm, Germany). Hexamethyldisilazane and trimethylchlorosilane were purchased from Fluka (Buchs, Switzerland) and used as supplied. Octadecylsilane (ODS)-bonded silica (preparative C 18 ; particle size, µm) was from Waters Associates Inc (Milford, MA). The material was washed with methanol, methanol/chloroform, 1:1 (vol/vol), methanol, and water before use. Triethylaminohydroxypropyl-Sephadex LH-20 (TEAP-LH-20) was synthesized and washed, 15 and before use, was converted into the bicarbonate form with CO 2. Activated silicic acid (Unisil, mesh) was from Clarkson Chemical Company (Williamsport, PA). Column beds were prepared in glass columns equipped with gauze-covered valves of teflon. All solvents were degassed before use. Suitable flow rates were obtained by application of nitrogen pressure. Liver Sample Collection and Cell Culture Conditions. In connection with a liver transplantation, human liver tissue was obtained from a 55-year-old female donor, who had died of intracranial bleeding. She had no history of liver disease, and all laboratory tests were normal. Hepatocytes were isolated and seeded on 60-mm dishes at a density of cells per dish as previously described. 9,12 The dishes were precoated with 0.2 ml of matrigel, a laminin-rich extracellular matrix prepared from Engelbreth-Holm-Swarm mouse sarcomas. 16 The cells were then incubated in Williams E medium (3 ml per dish) for 5 days. The medium, supplemented with glutamine (292 µg/ml), Na 2 SeO 3 (173 ng/ml), insulin (2 miu/ml), penicillin G sodium (100 U/mL), streptomycin sulfate (100 µg/ml), and gentamicin (85 µg/ml), was collected and renewed every 24 hours from the first to the fifth day of incubation, and on day 4, CsA was added to the medium (0-10 µmol/l). No dexamethasone or thyroid hormone was added to the cultures, because these hormones have a tendency to decrease rather than increase bile acid formation in human hepatocytes. 12 At the end of the incubation, cells were microscopically viable, and after collecting the medium, they were washed with 2 ml of phosphate-buffered saline and then removed in 1 ml of the buffer. The cell suspensions were combined (from 4 dishes), centrifuged at 500g (4 C), and after the supernatant had been aspirated, they were stored at 20 C until extraction. Separately, the variation of bile acid synthesis between cell cultures (6, each combined from 4 dishes before analysis) was determined. Expressed as the coefficient of variation, it was less than 25%. Approval to use parts of resected human liver specimens for research was given by the Ethics Committee at Huddinge University Hospital. Analytical Procedures. Following addition of isotopically labeled 27-hydroxycholesterol (260 pmol), CA (45,000 cpm), taurocholic acid (40,000 cpm ), and sulfated glycochenodeoxycholic acid (13,000 cpm) in 50 µl of ethanol to 8 or 11 ml of incubation medium (combined from 4 or 5 dishes, respectively), the medium was diluted with one volume of 0.5 mol/l aq. triethylamine sulfate (ph 7.2). Sterols and bile acids were then extracted on a column (15 8 mm) of ODS-bonded silica in water at 64 C. Under these conditions, steroid-protein interactions are minimized, permitting sorption of neutral and acidic steroids. The column was washed with 10 ml of water and 5 ml of 10% aq. methanol, before elution of steroids with 20 ml of 85% aq. methanol (eluting only a little cholesterol). 2 Group separation of neutral and acidic steroids was achieved by chromatography on a column (60 4 mm) of the lipophilic anion exchanger, TEAP-LH-20, packed in 85% aq. methanol. The eluate from the ODS-bonded silica column was passed through the anion exchanger, followed by a rinse with 5 ml of methanol, and these fractions containing the neutral steroids were combined. Retained acids were then eluted from the column in the following order: unconjugated C 27 - and C 24 -bile acids were eluted with 4 ml of 0.15 mol/l acetic acid in 95% aq. methanol; bile acids conjugated with glycine or taurine with 7 ml of 0.3 mol/l acetic acid ammonium acetate (apparent ph 6.6) in 95% aq. methanol; and sulfated bile acids with 13 ml of 0.5 mol/l potassium acetate potassium hydroxide (apparent ph 10.0) in 72% aq. methanol (sulfate fraction A). 15 The neutral steroid fraction from the TEAP-LH-20 column was taken to dryness in vacuo, and the residue was redissolved in 2 ml of dichloromethane/hexane, 4:1 (vol/vol). This solution was passed through a column (30 4 mm) of Unisil (packed in hexane and washed with 5 ml of dichloromethane/hexane, 4:1 [vol/vol]). The column was then washed with 10 ml of the same mixture (eluting cholesterol and other nonpolar sterols) before elution of oxysterols with 10 ml of ethyl acetate 14 and 6 ml of methanol (the latter fraction containing tetrahydroxysterols). Neutral steroids were then derivatized (see below). The unconjugated bile acid fraction was taken to dryness in vacuo, and the bile acids were derivatized (see below). The conjugated bile acid fraction contained bile acids conjugated with glycine or taurine, as well as monosulfates of neutral steroids. The solvent was removed in vacuo, and the residue (about 0.2 ml) was then diluted with 4 ml of water. The ph of the solutions was adjusted to 5.8 to 6.0, and 0.4 ml of 0.1 mol/l aq. sodium acetate (ph 5.6) was then added. Bile acid conjugates were then hydrolyzed by choloylglycine hydrolase (12 units) in the presence of 2-mercaptoethanol (20 µmol) and Titriplex III (20 µmol). 17 Following incubation for 16 hours at 37 C, the reaction mixture was extracted on a column (15 8 mm) of ODS-bonded silica in water. The column was washed with 5 ml of water before elution of bile acids with 8 ml of methanol. The eluate was passed through a column of TEAP-LH-20 (60 4 mm; packed in methanol), followed by a wash with 5 ml of methanol. Deconjugated bile acids were then eluted with 4 ml of 0.15 mol/l acetic acid in 95% aq. methanol and, after evaporation of the solvent, were derivatized. Monosulfates of neutral steroids were eluted from the column with 13 ml of 0.5 mol/l potassium acetate potassium hydroxide (ph 10) in 72% aq. methanol (sulfate fraction B). The sulfated bile acid fraction A contained bile acids conjugated with glycine or taurine and sulfuric acid, and also disulfates of neutral steroids if present. The ph of this fraction and of sulfate fraction B was adjusted to 7 with acetic acid, and the two fractions were combined. Following addition of 5 ml of water and removal of the alcohol in vacuo, the aqueous fraction (about 10 ml) was passed through a column (15 8 mm) of ODS-bonded silica (in water). The column was washed with 5 ml of water, and steroid sulfates were then eluted with 8 ml of methanol. The solvent was removed in vacuo and the residue was redissolved in 4 ml of water and 0.4 ml of 0.1 mol/l aq. sodium acetate (ph 5.6). Glycine and taurine conjugates were then enzymatically hydrolyzed, and the reaction mixture was extracted as described above for conjugated bile acids. The extract was taken to dryness in vacuo, and sulfate conjugates were then cleaved by solvolysis in 5 ml of tetrahydrofuran acidified with 5 µl of 4 mol/l sulfuric acid and heating at 50 C for 1 hour. 15 One milliliter of methanol was then added to the reaction mixture, and it was passed through a column (20 8 mm) of TEAP-LH-20 (packed in methanol and washed with 10 ml of tetrahydrofuran/ methanol, 5:1, vol/vol). The eluate and an additional rinse with 7 ml of tetrahydrofuran/methanol (5:1) contained desulfated neutral steroids. 15 This fraction was taken to dryness, the residue was redissolved in 1 ml of water, and the steroids were extracted with 2 1 ml of ethyl acetate before derivatization. Desulfated bile acids were also eluted from the column using 7 ml of 0.15 mol/l acetic acid in 95% aq. methanol, and, following removal of the solvent in vacuo, they were derivatized. The cells (about 0.2 ml) were extracted with 6 ml of 40% aq. ethanol in an ultrasonic bath for 15 minutes. Following centrifugation, the supernatant was transferred to a round-bottomed flask. The extraction procedure was repeated with 6 ml of ethanol, and to the combined extract were added the isotopically labeled steroids (see above). The solvent was removed in vacuo, and the residue was redissolved in 3 ml of methanol, followed by addition of 2 ml of water. Nonpolar steroids were then re-extracted by passing the solution through a column (15 8 mm) of ODS-bonded silica,

3 HEPATOLOGY Vol. 31, No. 6, 2000 AXELSON ET AL followed by 5 ml of water. The alcohol in the combined eluate (containing polar steroids) was removed in vacuo, and the aqueous residue (about 7 ml) was then re-extracted on the same column. The column was washed and steroids were eluted as described for steroids in medium (see above). Before gas-liquid chromatography (GLC) and gas chromatography mass spectrometry (GC/MS) analyses, all fractions were transferred with methanol to stoppered tubes and, following addition of 1.2 nmol triacontane and evaporation of the solvent, the steroids were derivatized. Bile acids were methylated with freshly prepared diazomethane at room temperature, using 1 ml of methanol/diethyl ether, 1:9 (vol/vol), as solvent. The solvent was taken to dryness after 30 minutes. Trimethylsilyl ethers of neutral and acidic steroids were prepared by addition of about 0.2 ml of pyridine/hexamethyldisilazane/ trimethylchlorosilane, 3:2:1 (by volume), and heating at 60 C for 30 minutes. The reagents were removed under a stream of nitrogen, and the derivatives were redissolved in 50 µl of hexane. GLC was performed using a Hewlett-Packard 5890 gas chromatograph connected to a Hewlett-Packard ChemStation. A fused silica column (25 m 0.32 mm) coated with a 0.17-µm layer of cross-linked methyl silicone (Ultra 1, Hewlett-Packard, Palo Alto, CA) was used with an on-column injection system and a flame ionization detector. The temperature of the oven was 50 C during the injection and, after 3 minutes, it was taken to 225 C at 35 C/min, and then programmed to 245 C at 0.3 C/min. After the injection, the pressure of the carrier gas (hydrogen) was increased from 6 to 13 psi. GC/MS was performed using a Finnigan SSQ 710 instrument housing a column identical to the one in the gas chromatograph ending in the ion source. An on-column injection device was used. The oven temperature was 50 C during the injection and, after 3 minutes, it was rapidly increased to 185 C, and then programmed to 280 C at a rate of 5 C/min. The electron energy was 50 ev, and repetitive scanning (30 scans/min) over the m/z range of 50 to 800 was started after a suitable delay. The identification of a steroid was based on the retention time and the complete mass spectrum, which were compared with those of the authentic steroid. The quantitation of a steroid was achieved by comparing its peak area in the gas chromatogram with that of the corresponding reference compound. The internal standard, triacontane, was used to normalize injection volumes. When concentrations of steroids were low and/or when other compounds interfered with the GLC analysis, quantitation was achieved by GC/MS. Quantitation was then based on the peak areas in selected ion chromatograms, which were compared with those given by known amounts of the corresponding reference substance. When the latter was not available, the amounts were estimated by converting selected ion current into total ion current and comparing the area of the total ion current peak with those given by reference compounds with analogous structure. RESULTS Identification of Steroids. The biosynthesis of bile acids by cultured primary human hepatocytes was investigated by using a general and analytically mild method for the isolation of oxysterols and bile acids from media and cells. Essentially, no major steroid was expected to escape detection. Recoveries of added isotopically labeled steroids after purification of samples were usually 75% to 85%. The final analysis was based on GLC and GC/MS. Following incubation of the hepatocytes, 17 steroids were identified in the media and cells (Tables 1 and 2). Neutral C 27 -steroids were mainly found in the cells and C 27 - and C 24 -bile acids in the medium. Sterols. In addition to cholesterol, 8 neutral sterols could be detected in the cell extracts, and 6 of these were also found in the media (Table 1, Fig. 1). With the exception of 5 -cholestane-3,7,12,27-tetrol, which was only detected No. TABLE 1. Sterols and Bile Acids in Media and Cells After Incubation of Human Hepatocytes Steroid Structure* Retention Index Molecular/Significant Ions (m/z) Sterols 1 C 5-3 -ol , 368, 329, 253, C 5-3,7 -ol 3110 (546), C 4-7 -ol-3-one , 457, 382, 269, C 5-3,7 -ol 3235 (546), C 4-7,12 -ol-3-one 3265 (560), 380, C 5-3 -ol-7-one , 382, 367, C 5-3,27-ol , 456, 417, C-3,7,12,27-ol 3450 (724), 544, 454, 343, C 4-7,12,27-ol-3-one 3600 (648), 558, 468, 267, 224 Bile acids B-3,7 -ol, 3155 (550), 460, 370, 355, B 5-3 -ol , 370, 331, 255, B-3,7 -ol 3195 (550), 370, 355, B-3,7,12 -ol 3210 (638), 458, 368, 343, B-7,12 -ol-3-one , 549, 474, 384, B 4-7,12 -ol-3-one 3345 (562), 382, 267, CA-3,7,12 -ol 3430 (680), 500, 410, 343, CA 4-7,12 -ol-3-one 3570 (604), 424, 267, 224 NOTE. GC/MS characteristics of identified sterols and bile acids as trimethylsilyl ethers and methyl ester trimethylsilyl ether derivatives, respectively, are shown. *C, cholestane; CA, cholestanoate; B, cholanoate; superscript indicates position of double bond; Greek letters denote configuration of hydrogen or hydroxyl groups. Kovats, on a fused silica capillary column coated with cross-linked methyl silicone. Molecular ion in parentheses is not seen in the mass spectrum ( 5% of base peak); base peak is shown in italics, intensities of fragment ions with m/z values above 200 to 300 were enhanced relative to those of lighter fragments; m/z, mass/charge. Only detected in cells. Only detected in the sulfate fractions. Tentative identification, reference bile acid not available. as a sulfate conjugate, all of these were present in the free form. A portion of 27-hydroxycholesterol was also sulfated. Most of the oxysterols found were potential bile acid precursors. 7 -Hydroxycholesterol, 7 -hydroxy-4-cholesten- 3-one, and 7,12 -dihydroxy-4-cholesten-3-one are all established intermediates in the classical or neutral pathway from cholesterol to bile acids. 1,2 However, expected 5 reduced forms of 7,12 -dihydroxy-4-cholesten-3-one could not be detected in spite of close examination. In contrast, its 27-hydroxylated derivative, 7,12,27-dihydroxy-4-cholesten-3-one, was found. 5 -Cholestane-3,7,12, 27-tetrol is a potential precursor of CA, but as mentioned above, it was exclusively found in a sulfated form. 27-Hydroxycholesterol was also detected, which is the first intermediate in the alternative or acidic pathway to bile acids, leading preferentially to CDCA. 2,18 In addition to these sterols, autooxidation products of cholesterol, such as 7 -hydroxycholesterol and 7-oxo-cholesterol, were present Hydroxycholesterol (see above) can also be formed by auto-oxidation. Bile Acids. The bile acid fractions contained 2 C 27 - and 6 C 24 -bile acids, the former group being unconjugated and the latter almost exclusively conjugated with glycine or taurine (Table 1). The major bile acids formed were CA and CDCA. In addition, 3 -hydroxy-5-cholenoic acid and 3,7 -

4 1308 AXELSON ET AL. HEPATOLOGY June 2000 TABLE 2. Daily Production of Bile Acids in Normal Human Hepatocytes No. Steroid Structure* Conjugation Amount of Steroid in Media After Incubation From: 0-24 h h h h (pmol/dish) 3-Oxy-steroids 4 C 5-3,7 -ol C 5-3 -ol-7-one C 5-3,27-ol B 5-3 -ol S Oxy-7 -hydroxy steroids 2 C 5-3,7 -ol C 4-7 -ol-3-one B-3,7 -ol G/T ,810 2, B-3,7 -ol S, ,480 2, B-3,7 -ol S Oxy-7,12 -dihydroxy steroids 8 5 -C-3,7,12,27-ol S CA 4-7,12 -ol-3-one CA-3,7,12 -ol B 4-7,12 -ol-3-one B 4-7,12 -ol-3-one G/T B-7,12 -ol-3-one G/T B-3,7,12 -ol B-3,7,12 -ol G/T 1,070 2,710 14,000 18, B-3,7,12 -ol S, Total (nmol/dish): Formation of Bile Acids. The production of bile acids by the hepatocytes was studied by determining the release of bile acids and their precursors into the medium during the first 4 days of incubation. The results are shown in Table 2. The production of bile acids was 1.8 mol per dish during the first day of incubation, and it increased rapidly to 25.4 nmol per dish on day 4. As shown, CA and CDCA conjugated with glycine or taurine were by far the major steroids, and on day 4, they accounted for as much as 73% and 23% of all steroids found, respectively. About half of the amount of CDCA also seemed to be conjugated with sulfuric acid. Only small amounts of sulfated 3 -hydroxy-5-cholenoic acid and 3,7 dihydroxy-5 -cholanoic acid were detected (about 2% of total). Potential bile acid precursors, such as unconjugated oxysterols, unconjugated C 27 - and C 24 -acids, and conjugated C 24 -acids, constituted only about 0.1%, 1%, and 2%, respectively. Trace amounts of sulfated sterols were also detected in NOTE. The amount of bile acids and their potential precursors were determined in media, collected with 24-hour intervals, during the incubation of human hepatocytes for 4 days. *C, cholestane; CA, cholestanoate; B, cholanoate. See also Table 1 for abbreviations. G/T, glycine or taurine conjugates; S, sulfate. Cells ( ; dish size, 28 cm 2 ) were incubated with 3 ml of Williams E medium. Media from 5 dishes were combined before the analysis. Can also be formed by auto-oxidation of cholesterol during purification of samples. Also conjugated with glycine or taurine. May include some of the bile acid conjugated only with glycine or taurine as a result of an incomplete separation. dihydroxy-5 -cholanoic acid were detected in the sulfate fractions. Whereas CA, CDCA, 3 -hydroxy-5-cholenoic acid, and 3,7 -dihydroxy-5 -cholanoic acid are considered to be metabolic end products, the remaining 4 C 27 - and C 24 -acids are all potential precursors of CA (they are 12 -hydroxylated). 3,7,12 -Trihydroxy-5 -cholestanoic acid (THCA) is an established precursor of CA, 1 but the acids possessing a 3-oxo group have not previously been considered as bile acid intermediates. However, when the neutral steroids are taken into account, they all formed a complete metabolic sequence from cholesterol to CA, as illustrated in Fig. 2. Other potential intermediates, including several of those required for the formation of CDCA, were absent or below the detection limit. However, CDCA precursors were found in a previous study on cultured human hepatocytes. 11 The precursors of CA observed in this study have previously been identified in media from cultured hepatoblastoma cells, 10 and some have also been identified in human blood. 10 FIG. 1. GC/MS analysis of oxysterols in normal human hepatocytes. Following incubation for 5 days in Williams E medium (the last day in the presence of 10 µmol/l CsA), the cells ( ) were taken for analysis by GC/MS. Fragment ion current chromatograms characteristic of trimethylsilyl ethers of oxysterols were constructed by the computer from mass spectra taken every 2 seconds during the analysis. For the purpose of illustration, the intensities of the ions (m/z) were multiplied by appropriate factors. The principal sterols indicated by the numbers are listed in Table 1. The equivalent of about cells was injected onto a Finnigan SSQ 710 instrument housing a fused silica column coated with methyl silicone.

5 HEPATOLOGY Vol. 31, No. 6, 2000 AXELSON ET AL the medium (0.1%), but the major portion of these was found in the cells (see Table 3). The daily release of CA and CDCA increased about 15 times (17-12) from day 1 to day 4. This was accompanied by a corresponding increase of their potential precursors, including 7 -hydroxy-4-cholesten-3-one and the acidic 12 hydroxylated intermediates, showing that they were formed by the cells. On day 4, 7,12 -dihydroxy-3-oxo-4-cholenoic acid and its 5 -reduced form were the major bile acid precursors detected. In contrast to these steroids, the release of 27-hydroxycholesterol rapidly decreased from day 1, when it was the major precursor, to day 4, when it was barely detected. The amounts of 7 -hydroxycholesterol remained relatively constant, which could be the result of a major contribution from auto-oxidized cholesterol (formed during the incubations or purification of samples). This contribution could be evaluated by comparing its amounts with those of 7 -hydroxycholesterol, which is usually produced to the same extent as 7 -hydroxycholesterol. 19 The distribution of bile acids and their precursors between the medium and cells is shown in Table 3. As expected, more than 95% (89%-99%) of the bile acids were present in medium. The only exception was 3 -hydroxy-5-cholenoic acid (as a sulfate), which was predominantly present in the cells. The cells also contained the major portion of free and sulfated neutral steroids. Table 3 also shows the effect of suppressing 27-hydroxylation in the hepatocytes by treating the cells with different concentrations of CsA. This drug is a potent inhibitor of the sterol 27-hydroxylase activity, particularly toward nonpolar substrates such as cholesterol, but not toward the polar substrate 5 -cholestane-3,7,12 -triol. 20,21 Whereas 1 µmol/l and 5 µmol/l had little or no effect on the production of bile acids, 10 µmol/l CsA decreased the production of CA by about 13% and that of CDCA by 30%. The amounts of the detected intermediates were characteristically changed during the treatment (Table 3). Thus, CsA treatment resulted in a 3-fold accumulation of 7 -hydroxy-4-cholesten-3-one and in a 10-fold accumulation of 7,12 -dihydroxy-4-cholesten-3- one (Fig. 1), but in decreased amounts of acidic CA precursors (including THCA) and of CA. It has previously been noted that CsA not only inhibits 27-hydroxylation, but also further conversion to a carboxyl group. 22 This may explain the observed 4-fold accumulation of 7,12,27-trihydroxy-4- cholesten-3-one. The results are consistent with a 27- hydroxylation of the intermediates, 7 -hydroxy-4-cholesten- 3-one and 7,12 -dihydroxy-4-cholesten-3-one, occurring in the hepatocytes. FIG. 2. A proposed biosynthetic pathway from cholesterol to CA in cultured normal human hepatocytes. The pathway is based on the occurrence of potential CA precursors in the incubation medium and cells, and their behavior under different experimental conditions. As seen, the origin of THCA is not known. DISCUSSION The development of techniques for preparing cultures of normal human hepatocytes offers new possibilities to study bile acid synthesis under experimental conditions. To evaluate the behavior of such cells and to obtain more information about biosynthetic pathways to bile acids in humans, the cellular accumulation and release of these compounds and their precursors have been investigated. The results show that isolated human hepatocytes in primary cultures behave as can be expected for intact liver cells, by converting almost completely large amounts of cholesterol into CA and CDCA and by conjugating them with glycine or taurine. However, the ratio between production rates of CA and CDCA was high (about 3:1). This may be a result of the absence of inhibitory bile acids in added medium, because this ratio is similar to those seen in patients with complete biliary drainage. 23 Furthermore, a relatively large portion of CDCA was sulfated, suggesting that sulfating enzymes may be more active in hepatocytes when cultured. Nonetheless, the high production of bile acids and the minute accumulation and release/leakage of bile acid precursors is in contrast to the behavior of cultured human hepatoblastoma cells (HepG2), previously used in studies on bile acid formation. 10 The latter cells release large amounts of bile acid precursors and unconjugated bile acids into the medium, and thus, it cannot be excluded that other aspects of bile acid synthesis, i.e., biosynthetic pathways, may be abnormal. CA and CDCA are formed from cholesterol via two major pathways, one starting with 7 -hydroxylation ( neutral pathway ) and the other starting with 27-hydroxylation ( acidic or alternative pathway ). 2,3 These pathways may be represented by the intermediates, 7 -hydroxycholesterol and 27- hydroxycholesterol, respectively. Because 7 -hydroxycholesterol can also be formed from cholesterol by auto-oxidation, its oxidized metabolite, 7 -hydroxy-4-cholesten-3-one, is a better marker for the neutral pathway. This has been demonstrated in several in vivo studies As shown in Table 2, the daily release of 7 -hydroxy-4-cholesten-3-one into the incubation medium increased in parallel to the production of bile acids. In contrast, the release of 27-hydroxycholesterol

6 1310 AXELSON ET AL. HEPATOLOGY June 2000 TABLE 3. Effects of CsA on the Production of Bile Acids in Normal Human Hepatocytes Amount of Steroid in Media and Cells After Incubation With CsA 0 mol/l 1 mol/l 5 mol/l 10 mol/l No. Steroid Structure* Conjugation Medium Cells Medium Cells Medium Cells Medium Cells (pmol/dish) 3-Oxy-steroids 4 C 5-3,7 -ol C 5-3 -ol-7-one C 5-3,27-ol C 5-3,27-ol S nd 18 nd 28 nd 23 nd 5 11 B 5-3 -ol S Oxy-7 -hydroxy steroids 2 C 5-3,7 -ol C 4-7 -ol-3-one B-3,7 -ol G/T 5, , , , B-3,7 -ol S, 2, , , , B-3,7 -ol S Oxy-7,12 -dihydroxy steroids 5 C 4-7,12 -ol-3-one nd 2 nd 2 nd 3 nd 21 9 C 4-7,12,27-ol-3-one nd 1 nd 1 nd 2 nd C-3,7,12,27-ol S CA 4-7,12 -ol-3-one ** CA-3,7,12 -ol ** 15 B 4-7,12 -ol-3-one 193 nd 125 nd 115 nd 60 ** 15 B 4-7,12 -ol-3-one G/T 285 nd 208 nd 310 nd 175 nd B-7,12 -ol-3-one G/T 143 nd 108 nd 200 nd 128 nd B-3,7,12 -ol 143 nd 88 nd 80 nd 58 ** B-3,7,12 -ol G/T 26, , , , B-3,7,12 -ol S, , , , Total (nmol/dish): NOTE. The amount of bile acids and their potential precursors was determined in media and cells after incubation with 0 to 10 µmol/l of CsA for 24 hours. CsA is an inhibitor of sterol 27-hydroxylase and was added to the media on the fourth day after the cells had been seeded. Abbreviation: nd, not detected. *C, cholestane; CA, cholestanoate; B, cholanoate. See also Table 1 for abbreviations. G/T, glycine or taurine conjugates; S, sulfate. Cells ( ; dish size, 28 cm 2 ) were incubated with 3 ml of Williams E medium in the presence or absence of CsA. Media from 4 dishes were combined before the analysis. Can also be formed by auto-oxidation of cholesterol during purification of samples. Also conjugated with glycine or taurine. May include some of the bile acid conjugated only with glycine or taurine as a result of an incomplete separation. **Unconjugated bile acid fraction lost. decreased. Assuming that the release of intermediates reflects their production, these results suggest that under the conditions used, the hepatocytes produced CA and CDCA mainly via the neutral pathway. This may seem surprising, because the acidic pathway is considered to be quantitatively important in vivo. 2,3 However, the hepatocytes were incubated in medium devoid of cholesterol and lipoproteins, and the decreasing amounts of 27-hydroxycholesterol is thus consistent with the hypothesis that cholesterol (and 27-hydroxycholesterol) used for the acidic pathway is mainly derived from lipoproteins in plasma. 2,10 The potential bile acid precursors identified in media and cells formed a complete metabolic sequence from cholesterol to CA, suggesting that they reflect a biosynthetic pathway (Fig. 2). This pathway starts with 7 -hydroxylation of cholesterol and oxidation to 7 -hydroxy-4-cholesten-3-one, followed by 12 -hydroxylation. Surprisingly, because all reactions in the steroid nucleus are generally believed to be completed before side-chain modifications, 1 the detected intermediates outline a different pathway to CA, in which 27-hydroxylation of the product, 7,12 -dihydroxy-4- cholesten-3-one, and further oxidation and cleavage of the side chain precede A-ring reduction. However, it must be pointed out that the mere occurrence of intermediates in media and cells only reveals a possible involvement in bile acid synthesis. The absence of intermediates does not exclude them from being formed, because they may be completely metabolized. In the present study, this was the case with many of the intermediates in the biosynthesis of CDCA. To obtain more information about the found intermediates, their behavior under various cell culture conditions was studied. Support for the outlined pathway shown in Fig. 2 was that the daily release into medium of the acidic intermediates with a 3-oxo group (12 -hydroxylated sterols could only be

7 HEPATOLOGY Vol. 31, No. 6, 2000 AXELSON ET AL detected in the cells) increased in parallel to those of 7 -hydroxy-4-cholesten-3-one and CA (Table 2). As expected, the release of the known CA precursor, THCA, also increased in a similar way. However, the origin of this compound could not be established in this study (see Fig. 2). It may be formed from 7,12 -dihydroxy-4-cholesten-3-one by A-ring reduction and then side-chain oxygenation, being the classical pathway to CA in humans, 1 but intermediates in such a pathway could not be detected. It is also possible that THCA is formed by A-ring reduction after 27-oxygenation of 7,12 -dihydroxy-4-cholesten-3-one, but before side-chain cleavage. 10 To determine if 7,12 -dihydroxy-4-cholesten-3-one could be 27-hydroxylated and converted to the acidic 3-oxo steroids, this reaction in the hepatocytes was suppressed by CsA. The resulting accumulation of the former steroid, accompanied with a decrease of the 3-oxo acids, provided strong evidence that this was the case (Table 3). Furthermore, a significant accumulation of 7,12 -dihydroxy-4-cholesten-3- one is only expected to be seen if this alternative pathway to CA is quantitatively important in the hepatocytes. For comparison, CsA treatment also resulted in an accumulation of 7 -hydroxy-4-cholesten-3-one (Table 3). This intermediate is known to be 27-hydroxylated in an analogous and major pathway to CDCA in the human liver. 1,3,7,11,27 However, intermediates to CDCA beyond 7 -hydroxy-4-cholesten-3- one, e.g.,7,27-dihydroxy-4-cholesten-3-one, 7 -hydroxy-3- oxo-4-cholestenoic acid, 2,10 and 5 -cholestane-3,7 -diol, were not observed in the present study. This was probably the result of a lower formation of CDCA than of CA by the cells. The production of CA and CDCA decreased by 6.3 nmol per dish during CsA treatment. Because these bile acids are mainly derived via the neutral pathway (see above) and the absolute amounts of accumulated intermediates were small (70 pmol/dish), their decreased production was probably caused by an inhibitory effect of CsA on cholesterol synthesis. 20 The alternative biosynthetic pathway to CA starting with 27-hydroxylation of 7,12 -dihydroxy-4-cholesten-3-one was observed here in isolated hepatocytes incubated in the absence of lipoproteins and bile acids. These conditions are expected to stimulate cholesterol and bile acid synthesis. Further studies will reveal how the pathway is affected by different incubation conditions. Furthermore, the hepatocytes used in this study were all derived from the same liver, but the formation of the key intermediate, 7,12 -dihydroxy- 3-oxo-4-cholenoic acid, has also been observed in human hepatocytes from other sources. Thus, the alternative pathway to CA does not seem to be limited to certain hepatocytes; in fact, it may also exist in the human liver. Support for this is that the intermediates, 7,12,27-trihydroxy-4-cholesten-3- one and 7,12 -dihydroxy-3-oxo-4-cholestenoic acid, are normal constituents in human blood, and that their levels seem to reflect the production of CA. 10 Furthermore, in patients with cerebrotendinous xanthomatosis, who lack the sterol 27-hydroxylase, 28,29 the hepatic accumulation of 7,12 dihydroxy-4-cholesten-3-one is greater than of other intermediates. 7 This can be expected if a significant portion of this intermediate is normally 27-hydroxylated, and it is in agreement with the present results on CsA-treated hepatocytes. Finally, it has been reported that in healthy humans, the major portion of CA is produced via a pathway that by-passes THCA. 30 The intermediates involved were not characterized, but may be those described here. 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