J. Biochem. 83, 527-535 (1978)
Formation of Bile Acids in Hemoglobin-Free Perfused Rat Livers
Michio OGURA, Midori GOTO, and Yoshikazu AYAKI Department of Biochemistry, Tottori University School of Medicine, Yonago, Tottori 683 Received for publication, August 16, 1977
The formation of bile acids was studied in isolated rat livers perfused with Krebs-Ringer bicarbonate buffer (pH 7.4)-5.5 DIM glucose oxygenated with OJCOt (95 : 5). When livers were perfused with 150 ml of the medium in a recirculating system, bile was secreted at a rate of around 30 mg/g liver/h during a 3-h period. Gas chromatographic analysis of biliary bile acid components gave the following results. As regards cholic acid, after that present in the hepatic pool was washed out during the initial 0.5 h, secretion of de novo cholic acid at a level of about 3 nmol/g liver/h lasted for at least 2 h. The pattern of bile acid was essentially similar to that in bile-fistula rats. However, secretion of /9-muricholic acid relative to cholic acid increased more than 60% in the perfusion period of 1.5-2.5 h compared with that in the period of 0.5-1.0 h. In tracer experiments with [24-14C]chenodeoxycholic acid, no accelerated incorporation into a- and /S-muricholic acids was observed in the perfusion period of 1.5-2.5 h, as compared with that of 0.5-1.5 h. Specific radioactivity of muricholic acids, especially of /9-muricholic acid, was far lower than that of chenodeoxycholic acid. These data suggest the existence of a compartmentalized pool of endogenous chenodeoxycholic acid.
The current knowledge of biosynthetic pathways or rabbits (7). Some of these investigations, -of bile acids has been obtained mainly by in vivo where [14C]cholesterol (2, 4, 6, 7) or "C-labelled •experiments with bile-fistula rats in early studies, precursors of cholesterol (6, 7) were used as and later by in vitro studies with rat-liver homog- tracers, showed that only small amounts of radio•enate or its subcellular fractions (7). Several activity were excreted in the bile or incorporated reports have also been published on the formation into biliary bile acids after infusion of the labelled •of bile acid by isolated perfused livers of rats (2-6) compounds into the perfusion medium. Furthermore, no parallel relation between the mass of bile acids excreted and the rate of incorporation Trivial names: Hyodeoxycholic, chenodeoxycholic, and of "C was observed (7). •deoxycholic acids; 3a,6ar-, 3ar,7a-, and 3a,\2a-4\Some investigators perfused rat livers with liydroxy-5£-cholan-24-oic acids. Cholic, a-muricholic, and ^-muricholic acids; 3ar,7ar,12a-, 3a,6£,7a-, and 3a, hemoglobin-free perfusion systems (8-13), but no •6£,7/?-trihydroxy-5/9-cholan-24-oic acids. analysis data on biliary bile acids in these livers Vol. 83, No. 2, 1978
527
M. OGURA, M. GOTO, and Y. AYAKI
528
have appeared. The present paper deals with the formation of bile acids in hemoglobin-free perfused rat livers. Although secretion of bile acids in these livers was similar in pattern to that in rats with a bile-fistula, a distinctive feature was an increase in the secretion of /5-muricholic acid relative to cholic acid in later periods of perfusion. No difference, however, was noted in the rate of transformation of exogenous [24-14C]chenodeoxycholic acid into aand /S-muricholic acids in livers during earlier and later periods of perfusion. From these and other results, the presence of a pool of endogenous chenodeoxycholic acid which serves as the immediate precursor of muricholic acids is discussed. MATERIALS AND METHODS Materials—Cholic acid and deoxycholic acid were obtained commercially and purified. Hyodeoxycholic acid was isolated from hog bile (14). Chenodeoxycholic acid, a-muricholic acid, and )S-muricholic acid were the same specimens described previously (15). Their purities were checked by melting point, specific optical rotation, thin-layer and gas chromatography. [24-uC]Chenodeoxycholic acid (50 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. It gave a single peak on a rad i oscan nogram. Other chemicals and organic solvents were of analytical reagent grade, the latter being distilled once prior to use. Perfusion Procedure—Perfusion apparatus: The apparatus was similar in principle to that originally described by Miller et al. (16), with some modifications. Figure 1 shows a diagram of the apparatus. It was designed for perfusing rat liver in either a nonrecirculating or a recirculating system. There were two sets of oxygenators and perfusion-medium reservoirs: one set (Ol and Rl) was utilized for perfusion in the nonrecirculating system and the other (O2 and R2), which included a mesh filter (F2) taken from a disposable blood transfusion set (Type 1, Terumo Co., Ltd., Tokyo), for that in the recirculating system. Perfusion medium was pumped by a roller pump (Type RPV3, Furue Science Co., Tokyo) with 2 channels (PI and P2) from the reservoirs to the respective
Fig. 1. Diagram of the perfusion apparatus. Outlines of the route through which the perfusion medium passed in the nonrecirculating and the recirculating systems are illustrated. The flow of gas mixture for oxygenation is omitted. For details, see the text. PI, P2, Rollar pump (2 channels); Rl, R2, reservoirs for perfusion medium; Ol, O2, oxygenators; MB, Mariotte bottle; Tl, T2, three-way stopcocks set at the position for perfusion in the nonrecirculating system; L, liver; V, liver-supporting vessel; S, device for measuring flow rate; Fl, F2, filters. oxygenators. A Mariotte bottle (MB) supplied the medium for the reservoir, Rl, keeping the level constant. The medium in the reservoir, R2, was continuously mixed by a magnetic stirrer. A gas mixture (O,/CO,, 95 : 5) was bubbled through the reservoirs and then passed through the oxygenators. The oxygenated medium from either of the two oxygenators passed through a three-way stopcock (Tl) into the liver (L) supported on a vessel (V) via a portal-vein cannula at a hydrostatic pressure of 20 cm. The perfusate leaving the liver passed through a cannula placed in the inferior vena cava into a graduated syringe (S). The level of the fluid in it gave an approximate measure of the flow rate through the liver by preliminary calibration. The perfusate passed through a filter (Fl) and reached another three-way stopcock (T2), which either passed the fluid out of the perfusion system or into the reservoir, R2. Thus, perfusion in either the nonrecirculating or the recirculating system could be achieved readily by setting the two stopcocks at suitable positions. The entire apparatus was housed in a cabinet J.
Biochem.
BILE ACIDS IN Hb-FREE PERFUSED LIVERS except for the Mariotte bottle, which was placed in an external water bath. The temperatures of the cabinet and of the bath were maintained at 37-38°C. The distal end of the biliary cannula was led out of the cabinet through a small window for collection of bile. Operating technique: Male Wistar strain rats, weighing 350-450 g, fed on a commercial rat diet (Oriental Kobo Kogyo Co., Osaka) were used in all experiments. They were fasted for 18 h. Operations for perfusing the livers were performed between 10:00 and 11:00 a.m. Each rat was anesthesized by intraperitoneal injection of Nembutal (5% solution of sodium pentobarbital; Abbott Laboratories, North Chicago, U.S.A.) at a dose of 0.1ml per 100 g body weight. The peritoneal cavity was opened and the bile duct was cannulated with polyethylene tubing No. 10 (Igarashi Ika Kogyo Co., Tokyo). The gastrohepatic ligament was cut and the esophagus was sectioned between two ligatures. The inferior vena cava was divided between two ligatures placed at a point distal to the liver and proximal to the right renal vein. The portal vein was then cannulated with a fine glass cannula, which was connected with the perfusion apparatus operating in the nonrecirculating mode and from which perfusion medium was continuously emerging. It was secured at the hilus by ligation. The portal vein was divided at a point closely distal to the cannula. The thorax was opened and a polyethylene tube was cannulated through the right atrium into the inferior vena cava, and tied firmly. The liver was then dissected out and placed in the supporting vessel of the perfusion apparatus with its diaphragmatic surface downwards. The vessel was covered with a thin sheet of polyethylene. The whole operation usually took 20 to 30 min. All perfusions both in the nonrecirculating and the recirculating systems were carried out at an approximate flow rate of 30 ml per min. Perfusion medium: In all experiments the perfusion medium was Krebs-Ringer bicarbonate buffer (pH 7.4) containing 5.5 mM glucose, which was prepared freshly and equilibrated with OJCO. (95 : 5). Livers were perfused with 150 ml of the medium in the recirculating system. In both the nonrecirculation and the recirculation techniques, the medium was pumped up to each oxygenator at a rate of 80 ml per min and the gas mixture was Vol. 83, No. 2, 1978
529
passed through it at a rate of 750 ml per min. Analysis of Bile Acids by Gas Chromatography —Bile (30-min portions) from 2 or 3 perfusions was pooled, and extracted with three 2-ml portions of hot ethanol. The extracts were filtered, combined and evaporated. The residue was partitioned between 8 ml each of ethanol/water ( 1 : 1 , v/v) and ether/n-heptane ( 1 : 1 , v/v) (77). The aqueous ethanol layer was evaporated to dryness and the residue was hydrolyzed in 12 ml of 2 N potassium hydroxide at 130°C for 4 h. The alkaline hydrolysate was diluted with 10 ml of water, and extracted with 20 ml of ether to remove unsaponifiable matter. After acidifying the alkaline aqueous layer with 6 N hydrochloric acid (Congo Red), bile acids were extracted three times with 30 ml portions of ether each. The ethereal extracts were combined, washed with water until neutral, dried over sodium sulfate, and evaporated. The residue dissolved in methanol/ether (1 :9, v/v) was methylated with a freshly prepared ethereal solution of diazomethane and the solvent was evaporated. The residue was then acetylated by heating with 1 ml of acetic anhydride at 140°C for 4 h and the reaction mixture was evaporated to dryness under a stream of nitrogen. The residue, containing acetyl derivatives of bile acid methyl esters, was dissolved in an appropriate amount of acetone and an aliquot of the solution was subjected to gas chromatographic analysis in a Shimadzu GC-3BF unit with a coiled glass column (3 mm i.d., 1.75 m long) packed with 0.75% SE-52 on Gas-Chrom Q (60-80 mesh). Injector and oven temperatures were 265°C and 235°C, respectively, and nitrogen was used as a carrier gas at a flow rate of 114ml/min. Alternatively, analysis was carried out with 3% OV-1 on Gas-Chrom Q (100-120 mesh), and in that case the injector and oven temperatures were 270°C and 240°C, respectively, and the flow rate of nitrogen was 78 ml/min. Quantitative determination was carried out by direct comparison of peak areas, measured by the triangulation method, with those obtained with the appropriate standards in a linear range. Metabolic Studies with [24-'4C]Chenodeoxycholic Acid—Perfusion of the labelled compound: Livers were perfused in the recirculating system. [24-"C]Chenodeoxycholic acid (1 [iC\, corresponding to 7.8 fg) dissolved in 0.5 ml of Krebs-Ringer
530
bicarbonate buffer (pH 7.4) as the sodium salt was injected directly into the perfusion medium just before it returned to the reservoir through a gummy tube fitted for this purpose. This infusion was carried out either 0.5 h or 1.5 h after the start of perfusion in the recirculating system. Samples of the perfusion medium were withdrawn for radioassay by a syringe through the gummy tube 5 min after infusion of the compound and then at selected intervals. Bile samples were collected in 30 min fractions. Analysis of isotopic metabolites: A 10-/il aliquot of each 30-min portion of bile collected after infusion of [24-14C]chenodeoxycholic acid was assayed for radioactivity. Remaining portions of bile samples were extracted with ethanol and the extracts were partitioned between two solvent phases as described above. One-fifth of the total bile acid fractions was chromatographed on a column (diameter, 0.6 cm) of 750 mg of silicic acid (100 mesh, Mallinckrodt Chemical Works, St. Louis, U.S.A.), and was eluted successively with water-saturated chloroform/ethanol [95 :5 (v/v), 5 ml; fraction L, discarded], water-saturated chloroform/ethanol/acetic acid/water [90 : 15 : 5 : 1 (by vol.), 10 ml; fraction I[, containing free bile acids and glycine-conjugates], and methanol/ acetic acid [100 : 1 (v/v), 3 ml; fraction III, containing taurine-conjugates] (Ozaki, K. et al., unpublished). Fractions II and III were radioassayed using one-tenth aliquots. Remaining portions of fraction II were rechromatographed on a similar column of silicic acid equilibrated with water-saturated chloroform/ ethanol (96 :4, v/v). Samples, dissolved in 5 ml of the same solvent, were applied to the column and eluted with 25 ml of water-saturated chloroform/ethanol ( 9 : 1 , v/v). The combined effluent contained free bile acids. Glycine conjugates were then eluted with 10 ml of water-saturated chloroform/ethanol/acetic acid/water (90 : 15 : 5 : 1, by vol.). These fractions were assayed for radioactivity. Remaining portions of total bile acid fractions were hydrolyzed and the acidic fraction was extracted as described above (cf. "Analysis of bile acids by gas chromatography"). Free bile acids were chromatographed on a column (diameter, 1.8 cm) of 5 g of silicic acid, being eluted successively with 100 ml each of chloroform/ethanol
M. OGURA, M. GOTO, and Y. AYAKI (v/v) [98 : 2 (fraction 1), 96 :4 (fraction 2), and 94 : 6 (fraction 3)] and then with 200 ml of the mixture (90 : 10, fraction 4) (18, 19). In this chromatography, monohydroxy bile acids, chenodeoxycholic, /9-muricholic, and a-muricholic acids were eluted in fractions 1, 2, 3, and 4, respectively. Each fraction was radioassayed using a onetwentieth aliquot. Remaining portions of these fractions from 3 perfusions were pooled and methylated with diazomethane. Methyl esters were chromatographed on thin-layer of plates Kieselgel H type 60 (E. Merck A.G., Darmstadt, Germany) with appropriate standards as references on both adjacent lanes. The plates were run up to 15 cm from the start in the following solvent systems: isooctane/ ethyl acetate/acetic acid (5 : 10 : 1, by vol.) for fraction 2, and methanol/acetone/chloroform (1 : 5 : 14, by vol.) for fractions 3 and 4. Fraction 4 was developed twice on the same plate. After compounds had been located with iodine vapor, plates were scanned for radioactivity on a thinlayer chromatogram scanner (Aloka, model TRM1B). The adsorbent in the zones corresponding to methyl esters of chenodeoxycholic acid (fraction 2), /9-muricholic acid (fraction 3), and a-muricholic acid (fraction 4) was scraped off the plate. Each was eluted from the gel and chromatographed on Sephadex-LH 20 as described previously (20). Collected effluents were evaporated to dryness and the residue was acetylated. Specific radioactivities of acetyl derivatives of the isolated bile acid methyl esters were determined by both radioassay and gas chromatographic analysis of suitable aliquots. Other Methods—Assay of protein in perfusates: Perfusates were filtered through a double layer of stocking nylon and filtrates were assayed for protein by the method of Lowry et al. (21), with a solution of bovine serum albumin, fraction V (Wako Pure Chemical Industries, Ltd., Osaka) as a standard. Hepatic oxygen consumption: The oxygen consumption in livers during perfusion in the nonrecirculating system was determined from the oxygen-tension values in the perfusate before and after passage through the liver (22), using an oxygen electrode calibrated with known gas mixtures. The flow rate through the liver was measured by collecting the venous effluent for a certain
J. Biochem.
531
BILE ACIDS IN Hb-FREE PERFUSED LIVERS
By gas chromatographic analysis, 6 bile acid components were detected in bile samples collected during various 30-min periods of perfusions. They were identified tentatively by comparison of their retention times with those of known bile acid standards (Table I). Their identities were further confirmed by gas chromatography-mass spectrometry using a 1 % OV-1 column in a Shimadzu GC-MS 7000 (the analysis was carried out at Kyoto Science Research Ltd., Kyoto). The mass spectrum of each component coincided with that of the appropriate standard. Figure 3 illustrates the secretion rates of cholic acid in perfused livers. The initial secretion rate of some 20 nmol/g liver/h dropped to a level of 3 to 4 nmol/g liver/h after a 0.5-h period of perfusion. RESULTS This level of secretion continued at least up to 2.5 h. Preliminary Perfusions in the Nonrecirculating In Fig. 4 amounts of bile acids secreted relative System—Some physiological parameters of livers to cholic acid are shown by direct comparison of perfused in the nonrecirculating system were gas chromatographic peak areas. Relative secremeasured during the initial 1 h. The results were tion of secondary bile acids, i.e., deoxycholic as well (mean±S.E.): oxygen consumption, 123 ±8.2 as hyodeoxycholic acid, decreased with time during /imol/g liver/h (n=6); bile secretion, 51.6±3.1 mg/g liver/h (n=6); protein production, 2.7±0.5mg/g TABLE I. Retention times of bile acid components liver/h (n=4). Bile Secretion and Biliary Bile Acids in Livers and reference compounds. Bile acids were analyzed Perfused in the Recirculating System—Livers were as their methyl ester acetates by gas chromatography perfused in the nonrecirculating system for a pre- with two stationary phases. Retention times are shown liminary 5 to 10-min period, and then perfusions relative to deoxycholic acid (=1.00). were switched to the recirculating system. Bile was R e I a t i v e retention t i m e s Bile acid component secreted at a level of around 30 mg/g liver/h or reference compound 7 3 % OV-1 throughout 3 h (Fig. 2).
period. Radioassay: Radioactivity of samples was counted in a liquid scintillation spectrometer (AJoka, model LSC 651). Quenching was corrected by automatic external standardization. Ten ml of liquid scintillator according to Bray (25) was used for the assay. For radioassay of the perfusate infused with [24-14C]chenodeoxycholic acid, a 0.5-ml aliquot was mixed with 2.5 ml of ethanol, and centrifuged at 2,000 rpm for 10 min. Two ml of the resulting supernatant was evaporated and the residue was dissolved in 10 ml of the scintillation fluid together with 0.2 ml of water and 0.5 ml of NCS tissue solubilizer (Amersham/Searle Co., Illinois, U.S.A.).
Pl»
.00
.00
Deoxycholic acid
.00"
.00°
.20
1.19
.18 .17
.38 .39
.30 .30
.52 .52
.47 .47
P2
Chenodeoxycholic acid P3
Cholic acid P4
Hyodeoxycholic acid
Fig. 2. Secretion of bile in perfused livers. Livers were perfused with Krebs-Ringer bicarbonate (pH 7.4)5.5 mM glucose in the recirculating system. Thirty-min portions of bile were collected. Results are expressed as means (±S.E.) of 6 perfusions. Vol. 83, No. 2, 1978
P5 ar-Muricholic acid
:>.O2 i!.O2
P6 /3-Muricholic add
;>.62 i2.65
'
.88 .88 2.46 2.46
a Figures denote numbers of peaks detected in the gas chromatogram. b Retention time at 9.9 min.
Formation of Bile Acids in Hemoglobin-Free Perfused Rat Livers
Michio OGURA, Midori GOTO, and Yoshikazu AYAKI Department of Biochemistry, Tottori University School of Medicine, Yonago, Tottori 683 Received for publication, August 16, 1977
The formation of bile acids was studied in isolated rat livers perfused with Krebs-Ringer bicarbonate buffer (pH 7.4)-5.5 DIM glucose oxygenated with OJCOt (95 : 5). When livers were perfused with 150 ml of the medium in a recirculating system, bile was secreted at a rate of around 30 mg/g liver/h during a 3-h period. Gas chromatographic analysis of biliary bile acid components gave the following results. As regards cholic acid, after that present in the hepatic pool was washed out during the initial 0.5 h, secretion of de novo cholic acid at a level of about 3 nmol/g liver/h lasted for at least 2 h. The pattern of bile acid was essentially similar to that in bile-fistula rats. However, secretion of /9-muricholic acid relative to cholic acid increased more than 60% in the perfusion period of 1.5-2.5 h compared with that in the period of 0.5-1.0 h. In tracer experiments with [24-14C]chenodeoxycholic acid, no accelerated incorporation into a- and /S-muricholic acids was observed in the perfusion period of 1.5-2.5 h, as compared with that of 0.5-1.5 h. Specific radioactivity of muricholic acids, especially of /9-muricholic acid, was far lower than that of chenodeoxycholic acid. These data suggest the existence of a compartmentalized pool of endogenous chenodeoxycholic acid.
The current knowledge of biosynthetic pathways or rabbits (7). Some of these investigations, -of bile acids has been obtained mainly by in vivo where [14C]cholesterol (2, 4, 6, 7) or "C-labelled •experiments with bile-fistula rats in early studies, precursors of cholesterol (6, 7) were used as and later by in vitro studies with rat-liver homog- tracers, showed that only small amounts of radio•enate or its subcellular fractions (7). Several activity were excreted in the bile or incorporated reports have also been published on the formation into biliary bile acids after infusion of the labelled •of bile acid by isolated perfused livers of rats (2-6) compounds into the perfusion medium. Furthermore, no parallel relation between the mass of bile acids excreted and the rate of incorporation Trivial names: Hyodeoxycholic, chenodeoxycholic, and of "C was observed (7). •deoxycholic acids; 3a,6ar-, 3ar,7a-, and 3a,\2a-4\Some investigators perfused rat livers with liydroxy-5£-cholan-24-oic acids. Cholic, a-muricholic, and ^-muricholic acids; 3ar,7ar,12a-, 3a,6£,7a-, and 3a, hemoglobin-free perfusion systems (8-13), but no •6£,7/?-trihydroxy-5/9-cholan-24-oic acids. analysis data on biliary bile acids in these livers Vol. 83, No. 2, 1978
527
M. OGURA, M. GOTO, and Y. AYAKI
528
have appeared. The present paper deals with the formation of bile acids in hemoglobin-free perfused rat livers. Although secretion of bile acids in these livers was similar in pattern to that in rats with a bile-fistula, a distinctive feature was an increase in the secretion of /5-muricholic acid relative to cholic acid in later periods of perfusion. No difference, however, was noted in the rate of transformation of exogenous [24-14C]chenodeoxycholic acid into aand /S-muricholic acids in livers during earlier and later periods of perfusion. From these and other results, the presence of a pool of endogenous chenodeoxycholic acid which serves as the immediate precursor of muricholic acids is discussed. MATERIALS AND METHODS Materials—Cholic acid and deoxycholic acid were obtained commercially and purified. Hyodeoxycholic acid was isolated from hog bile (14). Chenodeoxycholic acid, a-muricholic acid, and )S-muricholic acid were the same specimens described previously (15). Their purities were checked by melting point, specific optical rotation, thin-layer and gas chromatography. [24-uC]Chenodeoxycholic acid (50 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. It gave a single peak on a rad i oscan nogram. Other chemicals and organic solvents were of analytical reagent grade, the latter being distilled once prior to use. Perfusion Procedure—Perfusion apparatus: The apparatus was similar in principle to that originally described by Miller et al. (16), with some modifications. Figure 1 shows a diagram of the apparatus. It was designed for perfusing rat liver in either a nonrecirculating or a recirculating system. There were two sets of oxygenators and perfusion-medium reservoirs: one set (Ol and Rl) was utilized for perfusion in the nonrecirculating system and the other (O2 and R2), which included a mesh filter (F2) taken from a disposable blood transfusion set (Type 1, Terumo Co., Ltd., Tokyo), for that in the recirculating system. Perfusion medium was pumped by a roller pump (Type RPV3, Furue Science Co., Tokyo) with 2 channels (PI and P2) from the reservoirs to the respective
Fig. 1. Diagram of the perfusion apparatus. Outlines of the route through which the perfusion medium passed in the nonrecirculating and the recirculating systems are illustrated. The flow of gas mixture for oxygenation is omitted. For details, see the text. PI, P2, Rollar pump (2 channels); Rl, R2, reservoirs for perfusion medium; Ol, O2, oxygenators; MB, Mariotte bottle; Tl, T2, three-way stopcocks set at the position for perfusion in the nonrecirculating system; L, liver; V, liver-supporting vessel; S, device for measuring flow rate; Fl, F2, filters. oxygenators. A Mariotte bottle (MB) supplied the medium for the reservoir, Rl, keeping the level constant. The medium in the reservoir, R2, was continuously mixed by a magnetic stirrer. A gas mixture (O,/CO,, 95 : 5) was bubbled through the reservoirs and then passed through the oxygenators. The oxygenated medium from either of the two oxygenators passed through a three-way stopcock (Tl) into the liver (L) supported on a vessel (V) via a portal-vein cannula at a hydrostatic pressure of 20 cm. The perfusate leaving the liver passed through a cannula placed in the inferior vena cava into a graduated syringe (S). The level of the fluid in it gave an approximate measure of the flow rate through the liver by preliminary calibration. The perfusate passed through a filter (Fl) and reached another three-way stopcock (T2), which either passed the fluid out of the perfusion system or into the reservoir, R2. Thus, perfusion in either the nonrecirculating or the recirculating system could be achieved readily by setting the two stopcocks at suitable positions. The entire apparatus was housed in a cabinet J.
Biochem.
BILE ACIDS IN Hb-FREE PERFUSED LIVERS except for the Mariotte bottle, which was placed in an external water bath. The temperatures of the cabinet and of the bath were maintained at 37-38°C. The distal end of the biliary cannula was led out of the cabinet through a small window for collection of bile. Operating technique: Male Wistar strain rats, weighing 350-450 g, fed on a commercial rat diet (Oriental Kobo Kogyo Co., Osaka) were used in all experiments. They were fasted for 18 h. Operations for perfusing the livers were performed between 10:00 and 11:00 a.m. Each rat was anesthesized by intraperitoneal injection of Nembutal (5% solution of sodium pentobarbital; Abbott Laboratories, North Chicago, U.S.A.) at a dose of 0.1ml per 100 g body weight. The peritoneal cavity was opened and the bile duct was cannulated with polyethylene tubing No. 10 (Igarashi Ika Kogyo Co., Tokyo). The gastrohepatic ligament was cut and the esophagus was sectioned between two ligatures. The inferior vena cava was divided between two ligatures placed at a point distal to the liver and proximal to the right renal vein. The portal vein was then cannulated with a fine glass cannula, which was connected with the perfusion apparatus operating in the nonrecirculating mode and from which perfusion medium was continuously emerging. It was secured at the hilus by ligation. The portal vein was divided at a point closely distal to the cannula. The thorax was opened and a polyethylene tube was cannulated through the right atrium into the inferior vena cava, and tied firmly. The liver was then dissected out and placed in the supporting vessel of the perfusion apparatus with its diaphragmatic surface downwards. The vessel was covered with a thin sheet of polyethylene. The whole operation usually took 20 to 30 min. All perfusions both in the nonrecirculating and the recirculating systems were carried out at an approximate flow rate of 30 ml per min. Perfusion medium: In all experiments the perfusion medium was Krebs-Ringer bicarbonate buffer (pH 7.4) containing 5.5 mM glucose, which was prepared freshly and equilibrated with OJCO. (95 : 5). Livers were perfused with 150 ml of the medium in the recirculating system. In both the nonrecirculation and the recirculation techniques, the medium was pumped up to each oxygenator at a rate of 80 ml per min and the gas mixture was Vol. 83, No. 2, 1978
529
passed through it at a rate of 750 ml per min. Analysis of Bile Acids by Gas Chromatography —Bile (30-min portions) from 2 or 3 perfusions was pooled, and extracted with three 2-ml portions of hot ethanol. The extracts were filtered, combined and evaporated. The residue was partitioned between 8 ml each of ethanol/water ( 1 : 1 , v/v) and ether/n-heptane ( 1 : 1 , v/v) (77). The aqueous ethanol layer was evaporated to dryness and the residue was hydrolyzed in 12 ml of 2 N potassium hydroxide at 130°C for 4 h. The alkaline hydrolysate was diluted with 10 ml of water, and extracted with 20 ml of ether to remove unsaponifiable matter. After acidifying the alkaline aqueous layer with 6 N hydrochloric acid (Congo Red), bile acids were extracted three times with 30 ml portions of ether each. The ethereal extracts were combined, washed with water until neutral, dried over sodium sulfate, and evaporated. The residue dissolved in methanol/ether (1 :9, v/v) was methylated with a freshly prepared ethereal solution of diazomethane and the solvent was evaporated. The residue was then acetylated by heating with 1 ml of acetic anhydride at 140°C for 4 h and the reaction mixture was evaporated to dryness under a stream of nitrogen. The residue, containing acetyl derivatives of bile acid methyl esters, was dissolved in an appropriate amount of acetone and an aliquot of the solution was subjected to gas chromatographic analysis in a Shimadzu GC-3BF unit with a coiled glass column (3 mm i.d., 1.75 m long) packed with 0.75% SE-52 on Gas-Chrom Q (60-80 mesh). Injector and oven temperatures were 265°C and 235°C, respectively, and nitrogen was used as a carrier gas at a flow rate of 114ml/min. Alternatively, analysis was carried out with 3% OV-1 on Gas-Chrom Q (100-120 mesh), and in that case the injector and oven temperatures were 270°C and 240°C, respectively, and the flow rate of nitrogen was 78 ml/min. Quantitative determination was carried out by direct comparison of peak areas, measured by the triangulation method, with those obtained with the appropriate standards in a linear range. Metabolic Studies with [24-'4C]Chenodeoxycholic Acid—Perfusion of the labelled compound: Livers were perfused in the recirculating system. [24-"C]Chenodeoxycholic acid (1 [iC\, corresponding to 7.8 fg) dissolved in 0.5 ml of Krebs-Ringer
530
bicarbonate buffer (pH 7.4) as the sodium salt was injected directly into the perfusion medium just before it returned to the reservoir through a gummy tube fitted for this purpose. This infusion was carried out either 0.5 h or 1.5 h after the start of perfusion in the recirculating system. Samples of the perfusion medium were withdrawn for radioassay by a syringe through the gummy tube 5 min after infusion of the compound and then at selected intervals. Bile samples were collected in 30 min fractions. Analysis of isotopic metabolites: A 10-/il aliquot of each 30-min portion of bile collected after infusion of [24-14C]chenodeoxycholic acid was assayed for radioactivity. Remaining portions of bile samples were extracted with ethanol and the extracts were partitioned between two solvent phases as described above. One-fifth of the total bile acid fractions was chromatographed on a column (diameter, 0.6 cm) of 750 mg of silicic acid (100 mesh, Mallinckrodt Chemical Works, St. Louis, U.S.A.), and was eluted successively with water-saturated chloroform/ethanol [95 :5 (v/v), 5 ml; fraction L, discarded], water-saturated chloroform/ethanol/acetic acid/water [90 : 15 : 5 : 1 (by vol.), 10 ml; fraction I[, containing free bile acids and glycine-conjugates], and methanol/ acetic acid [100 : 1 (v/v), 3 ml; fraction III, containing taurine-conjugates] (Ozaki, K. et al., unpublished). Fractions II and III were radioassayed using one-tenth aliquots. Remaining portions of fraction II were rechromatographed on a similar column of silicic acid equilibrated with water-saturated chloroform/ ethanol (96 :4, v/v). Samples, dissolved in 5 ml of the same solvent, were applied to the column and eluted with 25 ml of water-saturated chloroform/ethanol ( 9 : 1 , v/v). The combined effluent contained free bile acids. Glycine conjugates were then eluted with 10 ml of water-saturated chloroform/ethanol/acetic acid/water (90 : 15 : 5 : 1, by vol.). These fractions were assayed for radioactivity. Remaining portions of total bile acid fractions were hydrolyzed and the acidic fraction was extracted as described above (cf. "Analysis of bile acids by gas chromatography"). Free bile acids were chromatographed on a column (diameter, 1.8 cm) of 5 g of silicic acid, being eluted successively with 100 ml each of chloroform/ethanol
M. OGURA, M. GOTO, and Y. AYAKI (v/v) [98 : 2 (fraction 1), 96 :4 (fraction 2), and 94 : 6 (fraction 3)] and then with 200 ml of the mixture (90 : 10, fraction 4) (18, 19). In this chromatography, monohydroxy bile acids, chenodeoxycholic, /9-muricholic, and a-muricholic acids were eluted in fractions 1, 2, 3, and 4, respectively. Each fraction was radioassayed using a onetwentieth aliquot. Remaining portions of these fractions from 3 perfusions were pooled and methylated with diazomethane. Methyl esters were chromatographed on thin-layer of plates Kieselgel H type 60 (E. Merck A.G., Darmstadt, Germany) with appropriate standards as references on both adjacent lanes. The plates were run up to 15 cm from the start in the following solvent systems: isooctane/ ethyl acetate/acetic acid (5 : 10 : 1, by vol.) for fraction 2, and methanol/acetone/chloroform (1 : 5 : 14, by vol.) for fractions 3 and 4. Fraction 4 was developed twice on the same plate. After compounds had been located with iodine vapor, plates were scanned for radioactivity on a thinlayer chromatogram scanner (Aloka, model TRM1B). The adsorbent in the zones corresponding to methyl esters of chenodeoxycholic acid (fraction 2), /9-muricholic acid (fraction 3), and a-muricholic acid (fraction 4) was scraped off the plate. Each was eluted from the gel and chromatographed on Sephadex-LH 20 as described previously (20). Collected effluents were evaporated to dryness and the residue was acetylated. Specific radioactivities of acetyl derivatives of the isolated bile acid methyl esters were determined by both radioassay and gas chromatographic analysis of suitable aliquots. Other Methods—Assay of protein in perfusates: Perfusates were filtered through a double layer of stocking nylon and filtrates were assayed for protein by the method of Lowry et al. (21), with a solution of bovine serum albumin, fraction V (Wako Pure Chemical Industries, Ltd., Osaka) as a standard. Hepatic oxygen consumption: The oxygen consumption in livers during perfusion in the nonrecirculating system was determined from the oxygen-tension values in the perfusate before and after passage through the liver (22), using an oxygen electrode calibrated with known gas mixtures. The flow rate through the liver was measured by collecting the venous effluent for a certain
J. Biochem.
531
BILE ACIDS IN Hb-FREE PERFUSED LIVERS
By gas chromatographic analysis, 6 bile acid components were detected in bile samples collected during various 30-min periods of perfusions. They were identified tentatively by comparison of their retention times with those of known bile acid standards (Table I). Their identities were further confirmed by gas chromatography-mass spectrometry using a 1 % OV-1 column in a Shimadzu GC-MS 7000 (the analysis was carried out at Kyoto Science Research Ltd., Kyoto). The mass spectrum of each component coincided with that of the appropriate standard. Figure 3 illustrates the secretion rates of cholic acid in perfused livers. The initial secretion rate of some 20 nmol/g liver/h dropped to a level of 3 to 4 nmol/g liver/h after a 0.5-h period of perfusion. RESULTS This level of secretion continued at least up to 2.5 h. Preliminary Perfusions in the Nonrecirculating In Fig. 4 amounts of bile acids secreted relative System—Some physiological parameters of livers to cholic acid are shown by direct comparison of perfused in the nonrecirculating system were gas chromatographic peak areas. Relative secremeasured during the initial 1 h. The results were tion of secondary bile acids, i.e., deoxycholic as well (mean±S.E.): oxygen consumption, 123 ±8.2 as hyodeoxycholic acid, decreased with time during /imol/g liver/h (n=6); bile secretion, 51.6±3.1 mg/g liver/h (n=6); protein production, 2.7±0.5mg/g TABLE I. Retention times of bile acid components liver/h (n=4). Bile Secretion and Biliary Bile Acids in Livers and reference compounds. Bile acids were analyzed Perfused in the Recirculating System—Livers were as their methyl ester acetates by gas chromatography perfused in the nonrecirculating system for a pre- with two stationary phases. Retention times are shown liminary 5 to 10-min period, and then perfusions relative to deoxycholic acid (=1.00). were switched to the recirculating system. Bile was R e I a t i v e retention t i m e s Bile acid component secreted at a level of around 30 mg/g liver/h or reference compound 7 3 % OV-1 throughout 3 h (Fig. 2).
period. Radioassay: Radioactivity of samples was counted in a liquid scintillation spectrometer (AJoka, model LSC 651). Quenching was corrected by automatic external standardization. Ten ml of liquid scintillator according to Bray (25) was used for the assay. For radioassay of the perfusate infused with [24-14C]chenodeoxycholic acid, a 0.5-ml aliquot was mixed with 2.5 ml of ethanol, and centrifuged at 2,000 rpm for 10 min. Two ml of the resulting supernatant was evaporated and the residue was dissolved in 10 ml of the scintillation fluid together with 0.2 ml of water and 0.5 ml of NCS tissue solubilizer (Amersham/Searle Co., Illinois, U.S.A.).
Pl»
.00
.00
Deoxycholic acid
.00"
.00°
.20
1.19
.18 .17
.38 .39
.30 .30
.52 .52
.47 .47
P2
Chenodeoxycholic acid P3
Cholic acid P4
Hyodeoxycholic acid
Fig. 2. Secretion of bile in perfused livers. Livers were perfused with Krebs-Ringer bicarbonate (pH 7.4)5.5 mM glucose in the recirculating system. Thirty-min portions of bile were collected. Results are expressed as means (±S.E.) of 6 perfusions. Vol. 83, No. 2, 1978
P5 ar-Muricholic acid
:>.O2 i!.O2
P6 /3-Muricholic add
;>.62 i2.65
'
.88 .88 2.46 2.46
a Figures denote numbers of peaks detected in the gas chromatogram. b Retention time at 9.9 min.
