Enterohepatic circulation of scymnol sulfate in an elasmobranch, the little skate (Raja erinacea)

Gert Fricker1, Ralph Wössner2, Jürgen Drewe3, Ruth Fricker4, and James L. Boyer5

1 Institut für Pharmazeutische Technologie und Biopharmazie, D-69120 Heidelberg, Germany; 2 Novartis AG, 4002 Basel; 3 Medizinische Poliklinik und Division für Gastroenterologie, Kantonsspital, 4031 Basel, Switzerland; 4 Department of Anesthesiology, Allgemeines Krankenhaus,University Hospital, A-1000 Vienna, Austria; 5 Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The sulfated bile alcohol scymnol sulfate (ScyS), 3alpha ,7alpha ,12alpha ,24xi ,26,27-hexahydroxy-5beta -cholestane-26(27)-sulfate, is the major bile salt in bile of an elasmobranch, the little skate. To investigate hepatic transport of bile alcohols in skate liver, [3H]ScyS and a potential precursor, 3alpha ,7alpha ,12alpha -trihydroxy-5beta -cholestane (chtriol), were used as model compounds. Their transport into isolated hepatocytes was partially saturable, temperature sensitive, and Na+ independent. The uptake of ScyS was inhibited by cholyltaurine, and uptake of cholyltaurine was inhibited by ScyS in a competitive manner. In contrast, uptake of chtriol was not inhibited by cholyltaurine, suggesting separate transport systems. ScyS and chtriol showed a choleretic effect in isolated perfused livers. When ScyS was added to the perfusate of isolated perfused livers, >25% was found in bile within 7 h. When chtriol was added to the perfusate, 10% of the dose was secreted into the bile mainly in the form of polar metabolites, whereas only nonmetabolized chtriol remained in the livers. The slow bile flow of 40-50 µl/h and the high recovery in the liver suggest that metabolism may be the rate-limiting step in the hepatic elimination of chtriol. The major metabolites secreted into bile were identified by mass spectrometry and chromatography as scymnol and ScyS. To study the enterohepatic circulation, [3H]ScyS or [3H]chtriol was administered into the duodenum of free-swimming skates, and bile was collected through exteriorized indwelling cannulas over a 4-day period. More than 90% of the radioactivity was recovered from bile, indicating that there was a highly effective absorption in the intestinal epithelium, as well as specific transport mechanisms for hepatic uptake and biliary secretion of these compounds. This is the first direct demonstration of an enterohepatic circulation for a bile alcohol sulfate in fish liver.

cholyltaurine; bile alcohol transport; bile acid transport; liver

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ALL VERTEBRATE LIVERS transport a wide variety of compounds from blood into bile. In most mammals, bile acids are the major solutes in bile. They are synthesized by the liver, efficiently excreted into the bile (21, 25, 29), and reabsorbed in the lower part of the small intestinal tract by active transport mechanisms (19, 22). Several carrier systems have been identified in the sinusoidal plasma membrane of rat hepatocytes, which transport bile acids from the portal blood (8, 9, 14, 26, 33), thus maintaining an efficient enterohepatic circulation. In some mammals, sulfated bile alcohols were also identified as major bile salts, e.g., tetra- or pentahydroxy alcohol sulfates in the bile of paenungulates (manatees, elephants) or some perissodactyla (rhinoceros, tapirs; Refs. 10, 20), and it may be assumed that they also undergo an enterohepatic circulation. There is very little information about the development of an enterohepatic circulation in lower vertebrates, but it seems to exist in reptiles and amphibians (17, 34). No data are available about an enterohepatic circulation in fishes. Although bile flow in marine elasmobranch vertebrates, such as the little skate (Raja erinacea), is ~100 times slower than in rodents (3), hepatobiliary transport has been shown to be the major pathway for the elimination of amphipathic organic anions, including cholyltaurine (3, 5, 6, 27). But, in many species of fish, bile acids are only minor components of bile, whereas sulfated bile alcohols are the major constituents (16, 32). Therefore, we studied the mechanisms underlying the hepatocellular uptake and secretion of a bile alcohol and its contribution to bile formation in isolated skate hepatocytes, isolated perfused skate liver, and free-swimming fish. As substrates (Fig. 1), we used scymnol sulfate [ScyS; 3alpha ,7alpha ,12alpha ,24xi ,26,27-hexahydroxy-5beta -cholestane-26(27)-sulfate], 3alpha ,7alpha ,12alpha -trihydroxy-5beta -cholestane (chtriol), which is an uncharged precursor of many physiological bile salts, and cholyltaurine.


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Fig. 1.   Substrates for investigation of bile salt transport in skate liver.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

All studies were performed at the Mount Desert Island Biological Laboratory in Salsbury Cove, ME.

Animals. Male skates (R. erinacea) with a mean body weight of 1.0 ± 0.3 kg were caught by local fishermen off Southwest Harbour, ME. The animals were maintained in aerated tanks supplied with continuously flowing seawater at 15°C and were used within 1-3 days of capture. Before hepatectomy, they were anesthetized with pentobarbital sodium (2.5 mg/kg), which was injected via a caudal vein.

Chemicals. Chtriol and [3H]chtriol with a specific radioactivity of 133 GBq/mmol were obtained from Prof. G. Kurz, University of Freiburg, Freiburg, Germany. ScyS was purified from skate bile by preparative thin-layer chromatography and characterized by mass spectrometry as descibed in detail previously (16). All other chemicals were purchased from commercial sources in the highest purity available.

Mass spectrometry. With the use of the method of electron ionization, mass spectra were made with a mass spectrometer MAT 312 (Finnegan, Bremen, Germany). Samples were dissolved in acetone and injected at a concentration of 10 µg/µl. The ionization energy was 70 eV, and the temperature of the ion source was 220°C. The spectra were analyzed with the Finnegan data system MAT SS 200.

High-performance liquid chromatography analyses. The high-performance liquid chromatography (HPLC) equipment consisted of two high-pressure pumps LKB-2150 (Pharmacia, Freiburg, Germany), a Rheodyne injector (Rheodyne, Cotati, CA), an ultraviolet detector rapid spectral detector 2140 (Pharmacia), and a flow-through scintillation counter Ramona 90 (Raytest, Straubenhardt, Germany). Bondapack C18 columns (3.9 mm × 300 mm; Waters, Eschborn, Germany) were used for analytic separation. All samples were diluted in the respective solvent and filtered through polyvinylidene difluoride filters with a pore size of 22 µm before injection.

Isolation of hepatocytes. Liver parenchymal cells were isolated by a modified collagenase perfusion technique, described in detail elsewhere (28). Briefly, the liver was removed from the abdominal cavity and was perfused via the portal vein at 15°C with Ca2+/Mg2+-free elasmobranch Ringer solution (in mM: 270 NaCl, 4 KCl, 3 MgCl2, 2.5 CaCl2, 0.5 Na2SO4, 1 KH2PO4, 8 NaHCO3, and 350 urea) before it was perfused with collagenase (0.07-0.1% in elasmobranch Ringer solution). Then, the liver was placed in 80 ml Ringer solution containing 100 units/ml deoxyribonuclease, and the cells were suspended subsequent to removal of the connective tissue capsule with a forceps. The cells were resuspended in elasmobranch Ringer solution to yield a final concentration of ~5-6 × 106 cells/ml. Most of the hepatocytes were obtained in clusters of three to six cells surrounding a single bile canaliculus, thus maintaining morphological polarity even in the isolated state. Only cells with a trypan blue exclusion >97% were used in transport studies. All kinetic experiments were performed within 2.5 h after cell isolation.

Transport studies with isolated cells. Freshly isolated hepatocytes (2.5-5 × 106 cells) were incubated at 15°C in 2.5 ml of elasmobranch Ringer solution containing various concentrations of radiolabeled and unlabeled chtriol, ScyS, or cholyltaurine. For inhibition experiments, the cell medium was supplemented with bile salts in concentrations as indicated in the legend to Fig. 3. The incubation flasks were gently agitated to minimize unstirred water layer effects and to keep the cells in homogeneous suspension. At the indicated time points, 500-µl portions of the suspension were removed and centrifuged in 1.5-ml polyethylene tubes in a Beckman microfuge at 12,000 g for 10 s. The supernatant was removed with a pasteur pipette, and the surface of the remaining pellet was washed with ice-cold elsamobranch Ringer solution and carefully blotted with filter tips to remove residual fluid on the surface. Substrate trapped in extracellular fluid was determined in control experiments by incubation of the cells with [14C]inulin and calculation of extracellular space in the pellet after centrifugation. The tubes were cut with a razor blade, and the tips containing the cells were transferred into scintillation vials filled with 250 µl of 4% sulfosalicylic acid. After 3-h incubation, the vials were vigorously shaken to disrupt the dispersed cells, and 5 ml of scintillation liquid (Optifluor; Packard Instruments, Downers Grove, IL) were added. The radioactivity was determined in a Packard Tri-Carb scintillation counter, by using the external standard ratio method to correct for quenching. Initial uptake rates were calculated from the cell-incorporated radioactivity, and curve analyses were performed with the data analysis program Enzfitter (Elsevier Publishers, Amsterdam, The Netherlands).

Isolated perfused skate liver. For the preparation of isolated skate livers, we followed a modification of previously described protocols (1, 24). Male skates were anesthesized by injection of 1% pentobarbital sodium (500 µl/kg body wt) into a tail vein. During surgery, the gills were superfused with 15°C seawater. The peritoneum was opened by a midline incision, and the bile duct was cannulated with a polyethylene tube. The portal vein was cannulated with another polyethylene tube, and the liver was perfused with Ca2+/Mg2+-free elasmobranch Ringer solution, containing 0.1% (wt/vol) heparin to prevent clotting. Then, the liver was carefully removed from all ligaments and transferred to a perfusion dish. The organ was first perfused at a flow rate of 30 ml/min in a nonrecirculating system with 200 ml complete elasmobranch Ringer solution. Then, the system was switched to a recirculation mode with 100 ml complete elasmobranch Ringer solution. All test compounds added were dissolved in dimethyl sulfoxide (final concentration 0.5% vol/vol) and injected within at least 1 min at the portal vein. Bile was collected at distinct time intervals in polyethylene tubes.

Sampling of radiolabeled ScyS. Radiolabeled ScyS was not available. Therefore, [3H]chtriol was administered by intravenous injection into the tail vein to free-swimming, bile duct-ligated little skates. Bile was collected by means of a balloon connected with a cannula, which had been inserted into the common bile duct of the fishes during a short pentobarbital sodium anesthesia. A plastic plug was placed in the gallbladder lumen to prevent bile from accumulating. The balloon was changed once every day over a 4-day period. More than 90% of the recovered radioactivity was ScyS, which was purified by HPLC.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of ScyS. ScyS was isolated from skate bile by preparative thin-layer chromatography. One milliliter of skate bile yielded on an average 100 mg ScyS, corresponding to a concentration of 18.2 mM in gallbladder bile. With the consideration of a total gallbladder bile volume of ~1.5 ml in the fish, the total ScyS pool was estimated to be ~27 µmol/fish.

Uptake of bile salts into freshly isolated skate hepatocytes. Uptake rates of chtriol, ScyS, and cholyltaurine (Fig. 1) into freshly isolated skate liver cells were determined as a function of increasing medium concentrations (0.1-10 µM chtriol and 5-100 µM ScyS or cholyltaurine, respectively) at an incubation temperature of 15°C. At all given concentrations, the amount of bile alcohols taken up by the cells increased linearily over at least 1.5 min, allowing the calculation of uptake rates by linear regression. The uptake of chtriol (Fig. 2, Table 1) and ScyS (Table 1) into isolated cells incubated in standard elasmobranch Ringer solution (containing 279 mM Na+) was not significantly different from uptake into cells incubated in an elasmobranch Ringer solution, where Na+ was replaced by choline+. The observed nonlinear relationship between flux rates of uptake and medium concentration indicates the presence of Na+-independent transport systems in addition to passive diffusion. Calculation of the kinetic parameters based on the assumption of a single transport system acting parallel to passive diffusion resulted in a flux rate (Jmax) of 134 ± 30 pmol · min-1 · mg-1, a Michaelis constant (KT) of 6.7 ± 3.1 µM, and a diffusion constant (KD) of 8.0 ± 1.6 µl · min-1 · mg-1 for chtriol and in a Jmax of 575 ± 25 pmol · min-1 · mg-1, a KT of 25 ± 12 µM, and a KD of 0.6 ± 0.2 µl · min-1 · mg-1 for ScyS. When the transport rates were determined at 4°C, uptake rates were diminished by 30%.


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Fig. 2.   Uptake rates (J) of 3alpha ,7alpha ,12alpha -trihydroxy-5beta -cholestane (chtriol) into freshly isolated skate hepatocytes as function of medium concentration (A). Cells were incubated either in standard elasmobranch Ringer solution (bullet ) or in a medium where Na+ salts were replaced by respective choline salts (open circle ). Line represents calculated saturable component, and dots show diffusional fraction of total uptake.

                              
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Table 1.   Uptake rates of chtriol and ScyS into isolated skate hepatocytes

Previous experiments have shown that bile acids are taken up by isolated skate heptocytes by a Na+-independent, saturable carrier-mediated transport mechanism (5, 6, 27). To clarify whether chtriol, ScyS, and cholyltaurine share common transport systems, the uptake of ScyS and chtriol was determined in the absence and presence of increasing concentrations of cholyltaurine. The uptake of ScyS was competitively inhibited by cholyltaurine (Fig. 3A). Vice versa, the uptake of cholyltaurine was competitively inhibited by ScyS with an inhibitory constant of 20 ± 6 µM (Fig. 3B). In contrast, no effect of 50-100 µM bile acid on the uptake rates of chtriol was seen (Fig. 3C), suggesting that bile acids and the uncharged alcohol do not share a common transport system for uptake into skate liver cells. Correspondingly, the uptake of cholyltaurine was noncompetitively inhibited by chtriol (Fig. 3D). This result confirms our recent findings demonstrating noncompetitive inhibition of bile salt uptake by uncharged bile alcohols (5).


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Fig. 3.   A: uptake of scymol sulfate (ScyS) in absence (open circle ) and presence of 100 µM cholyltaurine (bullet ). B: uptake of cholyltaurine in absence (open circle ) and presence of 50 µM ScyS (bullet ) and 100 µM ScyS (black-triangle). C: uptake of chtriol into freshly isolated hepatocytes in absence (open circle ) and presence of 50 µM cholyltaurine (bullet ) and 100 µM cholyltaurine (black-triangle). D: uptake of cholyltaurine in absence (open circle ) and presence of 25 µM chtriol (bullet ).

Isolated perfused skate liver. Bile production from isolated perfused skate livers averages ~2 µl · h-1 · g liver-1 or 40-50 µl/h (24). Therefore, bile has to be collected over relatively long time periods to obtain information about excretion or secretory maxima. [3H]chtriol was injected into the portal vein of isolated perfused skate livers, and bile was collected for 7 h. Figure 4A demonstrates the time course of secretion of radioactivity into bile after a bolus injection of 0.1 µM [3H]chtriol. The time points are corrected for the hepatic dead space volume of 2.9 µl/g liver according to Ref. 24 and for the dead space volume of the biliary cannula. The secretion of radioactivity reached a maximum at 3 ± 0.5 h after bolus injection. After 7.5 h, ~10% of the added radioactivity was recovered in the secreted bile, whereas 90% remained in the liver. Ten minutes after injection of [3H]chtriol, no radioactivity could be detected in the perfusate, suggesting a very rapid and complete uptake of [3H]chtriol into the liver. Binding to the tubing and glassware was neglegible. Over the time of maximum excretion, a slight transitory increase in the rate of bile flow could be observed (Fig. 4B).


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Fig. 4.   A: biliary excretion of radioactivity by isolated perfused skate liver after injection of 0.1 µM chtriol into portal vein. open circle , Cumulative excretion in percent of dose; bullet , excreted amount per time interval. B: bile flow of isolated perfused skate liver after injection of 0.1 µM chtriol into portal vein.

Because 90% of the added radioactivity was recovered from the liver, the organ was minced by means of scissors and further homogenized with a Potter-Elvejhem tissue grinder. Then, the homogenate was extracted with methanol/chloroform. Analysis of the extract by reverse-phase HPLC and comparison with reference chtriol (Fig. 5A) revealed that no metabolites had accumulated and that only intact chtriol was retained by the liver (Fig. 5B). Reverse-phase HPLC of bile samples indicated that only polar metabolites of chtriol were secreted into the bile, since unmetabolized chtriol was not found in the samples (Fig. 5C). The chromatogram exhibited one major metabolite, with a retardation factor (Rf) value corresponding to that of ScyS. It was purified by semipreparative C18 HPLC followed by absorption chromatography with a cleanup C18 cartridge. Analysis by negative-ion fast atom bombardment mass spectrometry showed one peak at a mass of 468 Da corresponding to the mass of scymnol (Fig. 6). Only a minor peak was found at a mass of 547 Da corresponding to the mass of ScyS. Treatment of ScyS in the same solvents as the metabolite indicated that under the conditions used, the sulfate residue is solvolized, resulting in the recovery of nonsulfated scymnol. These findings suggest that chtriol is a physiological precursor of scymnol and ScyS, respectively, in skate liver.


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Fig. 5.   A: reverse-phase HPLC chromatogram of chtriol. B: reverse-phase HPLC chromatogram of bile alcohol extracted from isolated perfused skate liver. C: reverse-phase HPLC chromatogram of bile alcohol metabolites excreted into bile of isolated perfused skate liver.


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Fig. 6.   Fast atom bombardment mass spectrum of major bile alcohol metabolite secreted into bile of isolated perfused skate liver. Relative mass of 468 Da is indicated for scymnol.

To study its hepatobiliary secretion, [3H]ScyS was also added to the perfusate of isolated perfused skate livers. The radioactivity was rapidly extracted from the circulating perfusate. Approximately 5 min after addition, radioactivity could be detected in bile (after correction for dead space), suggesting rapid and complete uptake into the liver. After 7.5 h of perfusion, >25% of the added radioactivity was recovered from bile (Fig. 7A). Only [3H]ScyS was found by HPLC, indicating that no further metabolism had occurred during the hepatic transit. The bile flow increased from a basal flow of 0.9 ± 0.2 to 1.5 ± 0.3 µl · h-1 · g liver-1 at the appearance of ScyS and remained at that level over 4 h. At the secretory maximum, the increase in bile flow (0.6 µl · h-1 · g liver-1 over basal flow) corresponded to 4.4 pmol · h-1 · g ScyS secreted-1. When the perfusion medium was supplemented with 50 µM ScyS, a significant choleretic effect was observed. Bile flow rates increased more than twofold within 15-30 min after addition of the sulfated bile alcohol into the perfusate (Fig. 7B).


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Fig. 7.   A: biliary excretion of radioactivity by isolated perfused skate liver after injection of 5 µM ScyS into portal vein. open circle , Cumulative excretion in percent of dose; bullet , excreted amount per time interval. B: bile flow of isolated perfused skate liver after injection of 50 µM chtriol into portal vein.

Enterohepatic circulation in the free-swimming skate. Both [3H]chtriol and [3H]ScyS were administered into the lumen of the gastrointestinal (GI) tract of free-swimming skates. For that purpose, animals were anesthetized, and the bile duct was cannulated. The cannula was exteriorized and connected to a small balloon fixed at the skin of the free-swimming fish, and bile was collected for up to 4 days. The test compound, diluted with 1 ml skate bile, was injected into the jejunum of the animal, ~1 cm below the papilla duodeni. At the end of the study, samples were collected from intestine, liver, and bile and analyzed for radioactivity. Table 2 summarizes the recovery rates. The data clearly demonstrate that the added compounds were nearly completely absorbed from the GI tract, taken up by the liver, and excreted into bile. Analysis of the biliary samples confirmed the complete transformation of chtriol to ScyS. The enteral administration of ScyS and the very high biliary recovery demonstrate for the first time an enterohepatic circulation of a sulfated bile alcohol in fishes.

                              
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Table 2.   Recovery of radioactivity after administration of chtriol and ScyS into the upper gastrointestinal tract of free-swimming skate

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study was performed to characterize the transport of bile alcohols by skate liver.

The most important finding was that the sulfated bile alcohol ScyS undergoes an efficient enterohepatic circulation in the little skate. Existence of an enterohepatic circulation has previously been shown for mammals, birds, and reptiles, all animals that possess C-27 or C-24 bile acids or taurine/glycine conjugates (11). Similar data in lower vertebrates like fishes have been lacking. The bile salts in cyclostomes and elasmobranches appear to be largely sulfated bile alcohols rather than bile acids (12), and ScyS has been shown to be the major bile salt in bile of several species of sharks and rays (16, 32). Our previous studies have demonstrated that bile acids are substrates for carrier-mediated transport in skate liver (3, 5, 6, 27). In contrast to bile acid transport in mammals, they appear to be taken up into hepatocytes by a single Na+-independent transport system (5, 6, 27). However, only small amounts of cholic acid have been found in the bile of skates and sharks (32), and it is likely that ScyS rather than bile acids is the end product of cholesterol metabolism in elasmobranches. The present studies provide evidence that chtriol is a physiological precursor of 3alpha ,7alpha ,12alpha ,24xi ,26,27-hexahydroxy-5beta -cholestane-26(27)-sulfate. Because it is metabolized to C-24 amidates in rodents, a transformation into a sulfated bile salt in skate liver was not unexpected. As indicated by the hepatic excretion of more polar metabolites, the hepatic permeation of chtriol occurs predominantly via a transcellular pathway. Both chtriol and ScyS are taken up by skate liver cells. Uptake of chtriol appears to occur by a saturable process, which is separate from the previously identified organic anion transport system recognizing bile acids. In contrast, ScyS and cholyltaurine seem to share the same uptake system. Because bile acids are only a minor component in elasmobranch bile, it may be assumed that bile acids, when given as exogenous substrates, use an organic anion transport system with a broader substrate specificity. Considering the structural similarity between cholyltaurine and ScyS, it is likely that this transport system may also represent the cellular uptake system for ScyS. Previously, a membrane polypeptide with an apparent molecular mass of 54,000 Da has been identified by photoaffinity labeling to be involved in Na+-independent cholyltaurine uptake into skate hepatocytes (6). This transport system remains to be characterized at the molecular level but seems not to share homology to the Na+-independent organic anion transporter cloned from rat and human liver (2, 13-15).

The biliary recovery of enterally administered ScyS in the free-swimming fish was >90%. At present, we do not know whether part of the sulfated alcohol is also eliminated via the urine of the fish. This may be the case, because a similar observation has been made in other species. Sulfated bile acids (3alpha - and 7alpha -sulfates) have been detected in both animal and human (18, 23), and it is known that such sulfated bile acids can be eliminated via the liver, but with a reduced biliary clearance (4, 7). Sulfation enhances the urinary excretion of such sulfated bile acids (30, 31).

In summary, ScyS, a sulfated bile alcohol, is taken up into skate hepatocytes by a Na+-independent, saturable transport system. The uncharged chtriol is a precursor of ScyS, which is secreted by the hepatocytes and efficiently reabsorbed from the skate intestine, providing the first evidence for an enterohepatic circulation in a marine species.

    ACKNOWLEDGEMENTS

We thank Prof. Kurz, University of Freiburg, Freiburg, Germany, for providing chtriol and David Hager, Doug Jutte, Alister Donald, and David Seward for excellent technical assistance.

    FOOTNOTES

This study was supported by the SmithKline Beecham Foundation Germany, the Lucille P. Markey Charitable Trust, North Atlantic Treaty Organization Collaborative Research Grant CRG-960281 (to G. Fricker), Mundipharma Switzerland, Mount Desert Island Biological Laboratory's Center for Membrane Toxicity Studies (National Institute of Environmental Health Sciences Grant P30-ES-03828), and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34989 and DK-25636 (to J. L. Boyer).

Address for reprint requests: G. Fricker, Institut für Pharmazeutische Technologie und Biopharmazie, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany.

Received 11 February 1997; accepted in final form 16 July 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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