Physicochemical and physiological properties of 5α-cyprinol sulfate, the toxic bile salt of cyprinid fish
2003; Elsevier BV; Volume: 44; Issue: 9 Linguagem: Inglês
10.1194/jlr.m300155-jlr200
ISSN1539-7262
AutoresTakaaki Goto, F. Holzinger, L.R. Hagey, Carolina Cerré, H-T. Ton-Nu, C.D. Schteingart, J.H. Steinbach, Benjamin L. Shneider, Alan F. Hofmann,
Tópico(s)Moringa oleifera research and applications
Resumo5α-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5α-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was ∼4 mM. At concentrations >1 mM, 5α-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5α-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax ≅ 0.5 μmol/min/kg) as compared with taurocholate. 5α-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5α-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5α-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5α-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit.These studies indicate that 5α-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish. 5α-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5α-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was ∼4 mM. At concentrations >1 mM, 5α-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5α-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax ≅ 0.5 μmol/min/kg) as compared with taurocholate. 5α-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5α-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5α-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5α-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5α-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish. In vertebrates, cholesterol is eliminated by conversion to water-soluble amphipathic, functional molecules called bile salts. Bile salts can be divided into three classes based on side-chain structure: C27 bile alcohols, C27 bile acids, and C24 bile acids (1Hofmann A.F. Schteingart C.D. Hagey L.R. Species differences in bile acid metabolism.in: Paumgartner G. Beuers U. Bile Acids in Liver Diseases. Kluwer Academic Publishers, Boston, MA1995: 3-30Google Scholar). After their biosynthesis from cholesterol, bile alcohols and bile acids undergo "conjugation," a biotransformation step that renders them water soluble and membrane impermeable at physiological pH. Bile alcohols are conjugated by esterification of the terminal C-27 hydroxy group with sulfate, whereas bile acids are usually conjugated by N-acyl amidation of the terminal C-27 or C-24 carboxyl group with taurine or glycine (2Hofmann A.F. Bile acids.in: Arias I.M. Boyer J.L. Fausto N. Jakoby W.B. Schachter D.A. Shafritz D.A. The Liver: Biology and Pathobiology. Third Edition. Raven Press, New York1994: 677-718Google Scholar, 3Une M. Hoshita T. Natural occurrence and chemical synthesis of bile alcohols, higher bile acids, and short side chain bile acids.Hiroshima J. Med. Sci. 1994; 43: 37-67PubMed Google Scholar). The occurrence of C27 bile alcohol sulfates is widespread in nature. They are the dominant bile salts of ancient mammalian species (elephant, manatee, hyrax, and rhinoceros) (4Hagey L.R. Bile Acid Biodiversity in Vertebrates: Chemistry and Evolutionary Implication. University of California–San Diego, San Diego, CA1992Google Scholar). They are also the major biliary surfactants present in cartilaginous fish (sharks, rays, and skates), herbivorous bony fish (carp, arapima, and angelfish), and in some amphibians (salamanders and frogs) (3Une M. Hoshita T. Natural occurrence and chemical synthesis of bile alcohols, higher bile acids, and short side chain bile acids.Hiroshima J. Med. Sci. 1994; 43: 37-67PubMed Google Scholar, 5Haslewood G.A.D. The Biological Importance of Bile Salts. North-Holland Publishing Co., Amsterdam, The Netherlands1978Google Scholar). One of the common bile alcohols is 5α-cyprinol, a molecule with five hydroxy groups that was originally isolated from the bile of Cyprinus carpio, the Asiatic carp. Cyprinol was shown to have hydroxy groups at C-3, C-7, C-12, C-26, and C-27, based on the work of Hoshita, Magayoshi, and Kazuno (6Hoshita T. Nagayoshi S. Kazuno T. Stero-bile acids and bile alcohols LIV. Studies on the bile of carp.J. Biochem. 1963; 54: 369-373Crossref PubMed Scopus (19) Google Scholar) and Anderson, Briggs, and Haslewood (7Anderson I.G. Briggs T. Haslewood G.A.D. Comparative study of bile salts. 18. The chemistry of cyprinol.Biochem. J. 1964; 90: 303-308Crossref PubMed Scopus (11) Google Scholar). Confirmation of the structure of the sulfate ester of 5α-cyprinol by proton and 13C-NMR as well as mass spectrometry (MS) has been reported by Asakawa et al. (8Asakawa M. Noguchi T. Seto H. Furihata K. Fujikura K. Hashimoto K. Structure of the toxin isolated from carp (Cyprinus carpio) bile.Toxicon. 1990; 28: 1063-1069Crossref PubMed Scopus (19) Google Scholar) 7Chemical Abstracts has assigned the registry number 15066-41-8 to 5α-cyprinol sulfate. Its index name is Cholestane-3,7,12,26,27-pentol, hydrogen sulfate, (3α,5α,7α,12α)−. The assignment of the sulfate to C-27 versus C-26 is arbitrary.. The A/B ring juncture of cyprinol is 5α (A/B trans), whereas the structure of most C27 and C24 bile acids is 5β (A/B cis). It has become customary to add a 5α prefix to cyprinol to indicate clearly its 5α-A/B trans juncture, and thus distinguish it from 5β-cyprinol (A/B cis), which is present in other fish, such as the sturgeon (9Haslewood G.A.D. Tammar A.R. Comparative studies of bile salts. Bile salts of sturgeons (Acipenseridae) and of the paddlefish Polyodon spathula: a new partial synthesis of 5 beta cyprinol.Biochem. J. 1968; 108: 263-268Crossref PubMed Scopus (10) Google Scholar). The structure of 5α-cyprinol sulfate is shown in Fig. 1. Most natural bile acids are amphipathic, possessing a hydrophilic side and a hydrophobic side (10Roda A. Hofmann A.F. Mysels K.J. The influence of bile salt structure on self-association in aqueous solutions.J. Biol. Chem. 1983; 258: 6362-6370Abstract Full Text PDF PubMed Google Scholar). The amphipathic structure of bile acids is responsible for their chief physiological function, which is to enhance absorption of dietary lipids. The C24 bile acids readily form mixed micelles with fatty acids and monoglycerides, and such mixed micelles can in turn solubilize fat-soluble vitamins. Such solubilization greatly enhances diffusion of insoluble lipids to the enterocyte brush border (11Hofmann A.F. Mysels K.J. Bile salts as biological surfactants.Colloids Surf. 1988; 30: 145-173Crossref Scopus (165) Google Scholar). The physicochemical properties of C24 bile acids have been investigated extensively (12Carey M.C. Physical-chemical properties of bile acids and their salts.in: Danielsson H. Sjövall J. Sterols and Bile Acids. Elsevier, Amsterdam1985: 345-403Crossref Scopus (80) Google Scholar, 13Cabral D.J. Small D.M. Physical chemistry of bile.in: Schultz S.G. Forte J.G. Handbook of Physiology. Section 6. The Gastrointestinal System. American Physiological Society, Bethesda, MD1989: 621-662Google Scholar), but few studies have examined the physicochemical properties of C27 bile acids and C27 bile alcohols. We hypothesized that the micelle-forming and solubilization properties of 5α-cyprinol sulfate should be similar to those of taurocholate, a molecule with a similar topology, as shown in Fig. 2, and performed studies to test this hypothesis. We also performed limited physiological studies on its ileal and hepatic transport in rodents because of its known toxicity for mammals (14Chen C.F. Yen T.S. Chen W.Y. Chapman B.J. Munday K.A. The renal, cardiovascular and hemolytic actions in the rat of a toxic extract from the bile of the grass carp (Ctenopharyngodon idellus).Toxicon. 1984; 22: 433-439Crossref PubMed Scopus (21) Google Scholar, 15Mohri T. Tanaka Y. Fukamachi K. Horikawa K. Takahashi K. Inada Y. Yasumoto T. Cyprinol as water-soluble poisoning component of carp.J. Food Hyg. Soc. 1992; 33: 133-143Crossref Scopus (11) Google Scholar, 16Yeh Y.H. Wang D.Y. Deng J.F. Chen S.K. Hwang D.F. Short-term toxicity of grass carp bile powder, 5alpha-cyprinol and 5alpha-cyprinol sulfate in rats.Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2002; 131: 1-8Crossref PubMed Scopus (13) Google Scholar, 17Xuan B.H.N. Thi T.X.N. Nguyen S.T. Goldfarb D.S. Strokes M.B. Rabenou R.A. Icthyotoxic ARF after fish gallbladder ingestion: a large case series from Vietnam.Am. J. Kidney Dis. 2003; 41: 220-224Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), including humans [reviewed in (17Xuan B.H.N. Thi T.X.N. Nguyen S.T. Goldfarb D.S. Strokes M.B. Rabenou R.A. Icthyotoxic ARF after fish gallbladder ingestion: a large case series from Vietnam.Am. J. Kidney Dis. 2003; 41: 220-224Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar)]. Finally, we examined some properties of 5α-cyprinol, the bile alcohol moiety of 5α-cyprinol sulfate, in order to define the possible in vivo significance of bacterial hydrolysis (deconjugation) of the ester bond linking sulfate to the bile alcohol. Gallbladders of C. carpio were obtained from a local fish market and an aquaculture facility (Loy Fisheries, Provo, UT) and stored in isopropanol. 5α-Cyprinol sulfate was isolated from the isopropanol-soluble extract of carp gallbladders. The extract was subjected to flash chromatography using a 30 × 5 cm column packed to 21 cm with silica gel, 40 μm (Flash Chrom Pack, J. T. Baker, Phillipsburg, NJ). The column was packed in chloroform-methanol (80:20; v/v). A highly concentrated isopropanol extract of carp bile was layered at the top of the column. A stepwise gradient of methanol in chloroform (80:20, 500 ml; 75:25, 500 ml; 70:30, 1,000 ml; 65:35, 500 ml) was used to elute the 5α-cyprinol sulfate. Fractions were examined by thin-layer chromatography (TLC) using a solvent system for conjugated bile acids (18Hofmann A.F. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid.J. Lipid Res. 1962; 3: 127-128Abstract Full Text PDF Google Scholar). Fractions containing pure 5α-cyprinol sulfate (Rf 0.25) were pooled and taken to dryness on a rotary evaporator. The structure of 5α-cyprinol sulfate (5α-cholestane-3α,7α,12α, 26,27-pentol-27-sulfate) was confirmed by proton magnetic resonance spectroscopy. Proton 1H-NMR was carried out at 500 MHz in the Department of Chemistry, University of California, San Diego. The solvent was deuterated methanol, and chemical shifts are expressed in ppm relative to tetramethylsilane: 0.697 (s, 3H, Me-18), 0.793 (s, 3H, Me-19), 0.996 (d, 7.0 Hz, 3H, Me-21), 2.129 (tt, 12.5 Hz, 3.5 Hz, 1H, H-5), 3.540 ν and 3.566 ν (ABX, Jab 11.0 Hz, Jax 6.6 Hz, Jbx 5.6 Hz, 2H, H-27), 3.765 (d, 5.0 Hz, 1H, H-7), 3.928 (m, 1H, H-3), 3.960 (s, 1H, H-12), 3.985 ν and 4.015 ν (ABX, Jab 9.5 Hz, Jax 5.0 Hz, Jbx 6.5 Hz, 2H, H-26). 5α-Cyprinol sulfate was precipitated from the isopropanol extract of carp gallbladders by the addition of several volumes of ethyl acetate. The precipitate (1.4 g) was dissolved in 2,2′-dimethoxypropane-1 N HCl (7:1; v/v) and maintained at 37°C for 12 h (19Cantafora A. Angelico M. Attili A.F. Ercoli L. Capocaccia L. An improved gas-chromatographic method for the determination of sulfated and unsulfated bile acids in serum.Clin. Chim. Acta. 1979; 95: 501-508Crossref PubMed Scopus (22) Google Scholar), the procedure resulting in complete solvolysis of the 5α-cyprinol sulfate. Water (200 ml) and chloroform-methanol (2:1, v/v) (800 ml) were added. The chloroform phase was evaporated to dryness, giving impure 5α-cyprinol. This was purified by silica gel column chromatography using chloroform-methanol, with stepwise increases in the proportion of methanol. Fractions were examined by TLC (18Hofmann A.F. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid.J. Lipid Res. 1962; 3: 127-128Abstract Full Text PDF Google Scholar), and those containing pure 5α-cyprinol (Rf 0.66) were pooled to give 0.9 g of 5α-cyprinol that was pure by TLC. The molecular weight of 5α-cyprinol was confirmed by electrospray (ESI)-MS. The critical micellization temperature (CMT) (also termed Krafft point) is the temperature at which the solubility of the monomer reaches the critical micellization concentration (CMC). At this temperature, there is a phase change: insoluble, crystalline material dissolves and forms micelles. A 20 mM solution of 5α-cyprinol sulfate in water was kept at 4°C and observed daily for 4 days to see if 5α-cyprinol sulfate precipitated from solution. Bottles were prepared containing three bile salt concentrations (3 mM, 5 mM, and 10 mM). For the 5 mM and 10 mM concentrations, calcium was added in increasing concentrations (0.2 M, 0.5 M, 1.0 M, 2.0 M, and 3.0 M). For the most-dilute bile salt concentration, calcium was added at the following concentrations: 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.75 M. Solutions of the sodium salts of three other conjugated C24 bile acids (taurocholate, glycocholate, and glycodeoxycholate) were prepared similarly. All solutions/dispersions were kept at room temperature and examined daily in a dark room with a light beam to check for precipitation. The ion product was calculated as a Ca2+ × [bile salt]−2 with an activity coefficient of 0.3 used for Ca2+ (20Jones C. Hofmann A.F. Mysels K.J. Roda A. The effect of calcium and sodium ion concentration on the properties of dilute aqueous solutions of glycine conjugated bile salts.J. Colloid Interface Sci. 1986; 114: 452-470Crossref Scopus (35) Google Scholar). The midpoint between the bile salt concentration at which precipitation was not present and the lowest concentration at which it was present was used to calculate the ion product. An excess of 5α-cyprinol was dispersed in distilled water and stirred intermittently for 1 week. The suspension was then centrifuged (2,000 g for 10 minutes) to sediment the insoluble 5α-cyprinol. One milligram of 5α-cholestane-3α,7α,12α, 24, 27-pentol dissolved in isopropanol was added to the supernatant. An aliquot was taken to dryness, converted to per-trimethylsilyl ethers using hexamethydilsilazane-trimethylchlorosilane-pyridine (2:1:10; v/v/v) (Tri-Sil Reagent, Pierce, Rockford, IL), and analyzed by gas chromatography (GC). The concentration of 5α-cyprinol was calculated from the peak ratio. The CMC of 5α-cyprinol sulfate was determined in two ways. The first was based on the change in surface tension in relation to aqueous concentration using the maximum bubble pressure method. A commercial device (Sensadyne 6000 Tensiometer, Chem-Dyne Research Corp., Milwaukee, WI) was used. The device was calibrated with distilled water and methanol; a bubble frequency of 1 bubble/sec was used. Solutions of sodium taurochenodeoxycholate (chenodeoxycholyltaurine) and 5α-cyprinol sulfate were prepared (17 mM in bile salt, 137 mM NaCl), and the change in bubble pressure measured as the solutions were diluted progressively with 0.154 M NaCl (room temperature). The CMC was defined as the intersection of the two lines obtained by extrapolating the linear portions of the two curves obtained when surface tension was plotted against the logarithm of the bile salt concentration (10Roda A. Hofmann A.F. Mysels K.J. The influence of bile salt structure on self-association in aqueous solutions.J. Biol. Chem. 1983; 258: 6362-6370Abstract Full Text PDF PubMed Google Scholar). The CMC was also obtained by a dye solubilization technique using Orange OT (1-O-tolyl azo-2-naphthol), a water-insoluble, micelle-soluble dye. Solubilization of Orange OT occurs only when micelles are present, and the amount solubilized is directly proportional to the concentration of micelles. A line was drawn through the first three points obtained above the base line and extrapolated to the base line. The point of intersection of this line with the base line was defined as the CMC. Experiments were performed at the same time with sodium taurocholate to permit comparison of the CMC of a 5α-cyprinol sulfate with that of a bile acid having the same nuclear substituents. CMC values obtained by the maximum bubble pressure method and the dye solubilization technique are known to agree well (10Roda A. Hofmann A.F. Mysels K.J. The influence of bile salt structure on self-association in aqueous solutions.J. Biol. Chem. 1983; 258: 6362-6370Abstract Full Text PDF PubMed Google Scholar). Conjugated bile acid anions are known to associate cooperatively with amphiphilic, water-insoluble molecules such as monoglycerides, resulting in micelles being formed at concentrations well below the CMC of a simple bile acid solution. The CMC of 5α-cyprinol sulfate in the presence of monooleylglycerol as well as its solubilizing capacity for this monoglyceride was determined by turbidometry as previously described (21Hofmann A.F. The function of bile salts in fat absorption: the solvent properties of dilute micellar solutions of conjugated bile salts.Biochem. J. 1963; 89: 57-68Crossref PubMed Scopus (199) Google Scholar). Attempts to characterize hepatic transport and biotransformation of 5α-cyprinol sulfate in the anesthetized biliary fistula rat could not be performed because the intravenous infusion of 5α-cyprinol sulfate at the physiological rate of 1 μmol/min/kg (a rate that is physiological for hepatic transport of conjugated C24 bile acids in the rat) caused hemolysis, hemobilia, and death. Accordingly, all studies of hepatic transport and biotransformation were performed using the single-pass IPRL, as described in detail elsewhere (22Bolder U. Ton-Nu H-T. Schteingart C.D. Frick E. Hofmann A.F. Hepatocyte transport of bile acids and organic anions in endotoxemic rats: impaired uptake and secretion.Gastroenterology. 1997; 112: 214-225Abstract Full Text PDF PubMed Scopus (203) Google Scholar). In this preparation, the liver is perfused with an electrolyte solution saturated with oxygen. Compounds were infused for 20 min at a physiological rate (for natural conjugated bile acids in the rat) of 1 μmol/min/kg animal body weight (about 25 nmol/g liver/min) or at 4 μmol/min/kg. Bile was collected in 5 min pools for 60 min. Biliary secretion was determined gravimetrically, and bile salt output was estimated by the enzymatic method commonly used to measure bile acids (23Turley S.D. Dietschy J.M. Re-evaluation of the 3α-hydroxysteroid dehydrogenase assay for total bile acids in bile.J. Lipid Res. 1977; 18: 404-407PubMed Google Scholar). First-pass extraction was determined by measuring the bile salt concentration in the entering and leaving cannulae. Biotransformation of 5α-cyprinol sulfate and 5α-cyprinol was determined by a variety of chromatographic methods. These included TLC systems previously developed to characterize bile acid biotransformation (24Oude Elferink R.P.J. de Haan J. Lambert K.J. Hagey L.R. Hofmann A.F. Jansen P.L.M. Selective hepatobiliary transport of nordeoxycholate side chain conjugates in mutant rats with a canalicular transport defect.Hepatology. 1989; 9: 861-865Crossref PubMed Scopus (52) Google Scholar), HPLC using a system previously described for separation of conjugated bile acids (25Rossi S.S. Converse J.L. Hofmann A.F. High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids.J. Lipid Res. 1987; 28: 589-595Abstract Full Text PDF PubMed Google Scholar), GC-MS (26Hagey L.R. Odell D. Rossi S.S. Crombie D.L. Hofmann A.F. Biliary bile acid composition of the Physeteridae (sperm whales).Mar. Mamm. Sci. 1993; 9: 23-33Crossref Scopus (7) Google Scholar), and electrospray ionization (ESI)-MS. For GC-MS, unconjugated bile acids were isolated by ether extraction of acidified bile. GC-MS was performed after alkaline deconjugation (2N NaOH, 4 h at 130°C) to identify individual bile acids present in amidated form. GC-MS was also performed on eluates from TLC spots. ESI-MS was performed at the Department of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA. The instrument was a Hewlett-Packard HP 1100 MSD operated in the negative or positive mode. The HPLC column was removed and the injector output coupled directly to the ESI inlet. Samples (2 μl) were injected in a 90:10 methanol-water (v/v) mobile phase running at a flow rate of 0.35 ml/min. The fragmenter was set at 200 V and the capillary voltage set to 5,000 V. Chromatography was performed before and after solvolysis using dimethoxypropane-HCl (19Cantafora A. Angelico M. Attili A.F. Ercoli L. Capocaccia L. An improved gas-chromatographic method for the determination of sulfated and unsulfated bile acids in serum.Clin. Chim. Acta. 1979; 95: 501-508Crossref PubMed Scopus (22) Google Scholar) to identify sulfates. To test whether transport of 5α-cyprinol sulfate competed with taurocholate transport by the IPRL, an infusion of 5α-cyprinol sulfate from 20–40 min was superimposed upon a continuous (60 min) infusion of 24-[14C]taurocholate. Output of radioactivity was determined in pools collected every 5 min. Influence of the 5α-cyprinol sulfate infusion on taurocholate uptake was determined by measuring radioactivity in the entering and leaving cannulas. To test whether 5α-cyprinol was absorbed from the small intestine, it was perfused into the jejunum or the ileum of the anesthetized biliary fistula rat using a recirculating system perfused at 4 ml/min, a rapid perfusion rate that minimizes the unstirred layer effect (27Marcus S.N. Schteingart C.D. Marquez M.L. Hofmann A.F. Xia Y. Steinbach J.H. Ton-Nu H-T. Lillienau J. Angellotti M.A. Schmassmann A. Active absorption of conjugated bile acids in vivo: kinetic parameters and molecular specificity of the ileal transport system in the rat.Gastroenterology. 1991; 100: 212-221Abstract Full Text PDF PubMed Google Scholar). The perfusate contained 5α-cyprinol at 0.1 mM concentration and 24-[14C]cholic acid at 0.1 mM, the latter being an absorbable solute that would be quantitatively excreted in bile (in conjugated form). The perfusate contained 130 mM NaCl, 20 mM d-glucose, 1.2 mM Ca2+, and 25 mM tris buffer, as well as phenol red, a nonabsorbable dye, to check paracellular permeability. The jejunal perfusate was adjusted to pH 6.5, the ileal perfusate to pH 7.0. The intestinal segment was perfused for 60 min and bile was collected for 120 min in 10 min pools. A bile sample taken during the steady state of biliary secretion was analyzed for radioactivity as well as by GC-MS to calculate relative rates of absorption. Competition for taurocholate uptake by 5α-cyprinol sulfate and 5α-cyprinol was examined in COS-7 cells transiently transfected with asbt, the conjugated bile acid transporter present in the apical membrane of the ileal enterocyte (28Sun A.Q. Ananthanarayanan M. Soroka C.J. Thevananther S. Shneider B. Suchy F.J. Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells.Am. J. Physiol. 1998; 275: G1045-G1055PubMed Google Scholar). Three days later, sodium-dependent taurocholate uptake was determined by incubating transfected COS-7 cells with 1.0 μM [3H]taurocholate in 116 mM NaCl or choline chloride. 5α-Cyprinol sulfate or 5α-cyprinol was added to the incubation buffer at concentrations ranging from 25 μM to 100 μM. After incubating for 15 min at 37°C, the cells were washed three times with 1.0 ml of ice-cold choline containing incubation buffer and lysed with 0.5 ml Triton X-100 in water. Aliquots were taken to determine cell-associated protein and radioactivity. Four freshly killed carp were purchased at a Japanese fish market. Gallbladder contents were aspirated for enzymatic determination (23Turley S.D. Dietschy J.M. Re-evaluation of the 3α-hydroxysteroid dehydrogenase assay for total bile acids in bile.J. Lipid Res. 1977; 18: 404-407PubMed Google Scholar) of the concentration of 5α-cyprinol sulfate. Four other carp were fed and killed 4 h later. The concentration of 5α-cyprinol sulfate in the proximal intestine was determined enzymatically (23Turley S.D. Dietschy J.M. Re-evaluation of the 3α-hydroxysteroid dehydrogenase assay for total bile acids in bile.J. Lipid Res. 1977; 18: 404-407PubMed Google Scholar). Samples from both proximal and distal intestine were examined by TLC (18Hofmann A.F. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid.J. Lipid Res. 1962; 3: 127-128Abstract Full Text PDF Google Scholar) to assess whether 5α-cyprinol was present. By ESI-MS (negative mode), carp bile contained predominantly (95%) 5α-cyprinol sulfate and 5% of a compound having the molecular weight (515.4) of a C27 bile alcohol sulfate with four hydroxy groups. (Fig. 3). This compound is most likely 5α-cholestane-3α,7α,12α,26-tetrol (29Wang M.Y. Elliott W.H. Bile acids. LXXVII. Large-scale preparation of 5 alpha-anhydrocyprinol from carp bile.Prep. Biochem. 1985; 15: 191-209PubMed Google Scholar) esterified with sulfate at C-26 (29Wang M.Y. Elliott W.H. Bile acids. LXXVII. Large-scale preparation of 5 alpha-anhydrocyprinol from carp bile.Prep. Biochem. 1985; 15: 191-209PubMed Google Scholar). Bile acids were not present, although they have been reported to be present in some samples of carp bile (29Wang M.Y. Elliott W.H. Bile acids. LXXVII. Large-scale preparation of 5 alpha-anhydrocyprinol from carp bile.Prep. Biochem. 1985; 15: 191-209PubMed Google Scholar). ESI-MS in the positive mode indicated that phospholipids are not present in carp bile. By GC-MS, cholesterol was present in trace amounts. A solution of 5α-cyprinol sulfate was stable at 4°C, indicating that its CMT was below this temperature. The ion product of Ca2+ × [5α-cyprinol sulfate]−2 was about 9.5 × 10−7 M3. The calcium salt of 5α-cyprinol sulfate was less soluble than that of glycocholate and taurocholate, but more soluble than that of glycodeoxycholate, whose calcium salt has an ion product of 0.02 × 10−7 M3 (20Jones C. Hofmann A.F. Mysels K.J. Roda A. The effect of calcium and sodium ion concentration on the properties of dilute aqueous solutions of glycine conjugated bile salts.J. Colloid Interface Sci. 1986; 114: 452-470Crossref Scopus (35) Google Scholar). The aqueous solubility of 5α-cyprinol was 360 μM, a value similar to that of cholic acid (30Roda A. Fini A. Effect of nuclear hydroxy substituents on aqueous solubility and acidic strength of bile acids.Hepatology. 1984; 4: 72-76Crossref Scopus (44) Google Scholar). Thus 5α-cyprinol was poorly soluble and did not form micelles. Figure 4shows the relationship between surface tension and bile salt concentration for the sodium 5α-cyprinol sulfate and sodium taurochenodeoxycholate. The surface tension for each molecule decreased with increasing concentration. The calculated CMC value (see Methods) was about 1.54 mM; this concentration is probably a concentration at which aggregation of monomers begins when Na+ is present at 0.15 M. Figure 5shows the solubilization of Orange OT by the sodium 5α-cyprinol sulfate and sodium taurocholate. The CMC of 5α-cyprinol sulfate by this technique was about 4.5 mM; that of taurocholate was about 9 mM. Both values were obtained with a total [Na+] of 0.154 M. Figure 6shows the solubilization of monooleylglycerol by sodium 5α-cyprinol sulfate and sodium taurocholate. For 5α-cyprinol sulfate, monoolein solubilization began at 1–2 mM, and for taurocholate, at about 3.5 mM. The slope of the solubilization curve (Δ monoolein solubilized/Δ bile salt) was 2.1 for 5α-cyprinol sulfate, a value slightly greater than that observed for taurocholate, which was 1.8. 5α-cyprinol sulfate was infused at a rate of 1 μmol/min/kg for 20 min into the single-pass IPRL. TLC of bile showed two spots. The major spot had the mobility of unchanged 5α-cyprinol sulfate. A minor spot had a slower mobility, compatible with its being a disulfate of 5α-cyprinol sulfate; by its staining properties, it did not contain glucuronic acid. Both spots were eluted, subjected to solvolysis, and rechromatographed. For each spot, the solvolysis product had the mobility of 5α-cyprinol. Thus, 5α-cyprinol sulfate was transported without biotransformation except for a small fraction that underwent additional sulfation. Recovery of 5α-cyprinol sulfate at the infusion rate of 1 μmol/min/kg was incomplete (65.3 ± 10.2%, n = 6). At the infusion rate of 4 μmol/min/kg, recovery was extremely low (17.0%, mean of two experiments). Because 5α-cyprinol sulfate might undergo hydrolysis durin
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