Evidence for an anion exchange mechanism for uptake of conjugated bile acid from the rat jejunum

Andree Amelsberg, Christina Jochims, Claus Peter Richter, Rolf Nitsche, and Ulrich R. Fölsch

Department of Medicine, Christian-Albrechts University, 24105 Kiel, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Absorption of conjugated bile acids from the small intestine is very efficient. The mechanisms of jejunal absorption are not very well understood. The aim of this study was to clarify the mechanism of absorption of conjugated bile acid at the apical membrane of jejunal epithelial cells. Brush-border membrane vesicles from intestinal epithelial cells of the rat were prepared. Absorption of two taurine-conjugated bile acids that are representative of endogenous bile acids in many variate vertebrate species were studied. In ileal, but not jejunal brush-border membrane vesicles, transport of conjugated bile acids was cis-stimulated by sodium. Transport of conjugated bile acids was trans-stimulated by bicarbonate in the jejunum. Absorption of conjugated dihydroxy-bile acids was almost twice as fast as of trihydroxy-bile acids. Coincubation with other conjugated bile acids, bromosulfophthalein, and DIDS, as well as by incubation in the cold inhibited the transport rate effectively. Absorption of conjugated bile acids in the jejunum from the rat is driven by anion exchange and is most likely an antiport transport.

jejunal absorption; brush-border membrane vesicles


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CONJUGATED BILE ACIDS are secreted into the intestine during digestion. Bile acid absorption from the small and large intestine is efficient, with >90% of the bile acids secreted with a meal being conserved (9). As a consequence of this efficient absorption, a large pool of bile acids accumulates and circulates multiple times each day.

Conjugated bile acid uptake by the small intestine is mainly by carrier-mediated transport. Separate transporters appear to be present in the jejunum and ileum. In addition, a small fraction of the more lipophilic conjugates may be absorbed passively in protonated form if the pH of duodenal contents is sufficiently acidic (2). Conjugated bile acids may undergo deconjugation by bacterial enzymes in the small intestine before absorption (9). The liberated unconjugated bile acids, being weak acids, can be absorbed from the small intestine by passive mechanisms.

Active absorption of conjugated bile acids has long been known to involve a sodium-dependent transporter in the apical membrane of the ileal enterocyte. This transporter has been cloned from hamster (32), rat (25), and human ileum (33); has been shown to be present in the renal proximal tubule in the rat (5); and has been found to have homology with the sodium-dependent transporter for conjugated bile acids that is present in the basolateral membrane of the hepatocyte (7). Because resection of the ileum causes severe bile acid malabsorption, the ileal transporter was considered to be the major transporter responsible for the efficient intestinal absorption of conjugated bile acids.

Nonetheless, there is a large body of evidence indicating that conjugated bile acids are absorbed from the jejunum despite convincing evidence that the ileal transporter is absent in the jejunum, at least in several species. In humans, the evidence for jejunal absorption includes perfusion studies that have shown uptake of conjugated bile acids (4, 8, 30, 31), as well as measurements of bile acids in jejunal aspirates (3) and plasma (21) that suggested a more proximal and therefore earlier absorption of dihydroxy-conjugated bile acids. In the pig, diversion of intestinal content around the jejunum greatly decreases bile acid absorption, suggesting that the majority of conjugated bile acids occurs in the jejunum (12). In rats, estimates of jejunal absorption range from 12 to 60% of total bile acid absorption (17, 20, 27, 29). Evidence for proximal absorption of conjugated bile acids in nonmammals such as hens (10), pigeons (28), and turkeys (26) has been reported.

In an attempt to define the mechanism for jejunal absorption of conjugated bile acids, we performed jejunal perfusion studies in the guinea pig and found evidence for carrier-mediated transport based on saturability and competitive inhibition (2). The transport rate was greater for dihydroxy-conjugated bile acids than trihydroxy-conjugated bile acids in contrast to ileal transport, where trihydroxy-conjugates are transported more rapidly (18).

Perfusion studies do not permit assessment of ion dependency of transport and also do not clearly distinguish transcellular from paracellular absorption, even though paracellular absorption seems unlikely for large, charged molecules such as conjugated bile acid anions. In the present study, we have prepared isolated brush-border membranes from the rat to 1) gain further insight into the mechanism of jejunal transport of conjugated bile acid anions, 2) test whether a transport mechanism for conjugated bile acid anions is also present in the apical membrane of the rat jejunal enterocyte, and 3) determine whether the apical transporter of the rat enterocyte had a substrate affinity profile that was similar to that observed in the perfused guinea pig jejunum.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Male Sprague-Dawley rats (Harlan Winkelmann, Borchen, Germany) weighing 250-275 g were fed ad libitum and kept on a 12:12-h light-dark cycle for at least 1 wk before use. Food, but not water, was removed from the cage 24 h before conducting an experiment.

D-[U-14C]glucose (9.5-13.3 GBq/mmol) and cholyltaurine (C-tau), [G-3H]taurocholate (74-185 GBq/mmol), were purchased from DuPont NEN (Bad Homburg, Germany). Chenodeoxycholyltaurine (CDC-tau), 22-23-[3H]taurochenodeoxycholate, was a generous gift from Dr. Alan F. Hofmann (University of California, San Diego, CA). Adenosine triphosphate, lactate dehydrogenase, nicotine adenine nucleotide, phosphoenolpyruvate, and pyruvate kinase were obtained from Boehringer Mannheim. Nonradioactive bile acids [C-tau, CDC-tau, and deoxycholyltaurine (DC-tau)], ouabain, dithiothreitol, D-glucose, D-mannitol, DMEM, EDTA, and EGTA were bought from Sigma-Aldrich (Deisenhof, Germany). All other reagents were of analytic grade and were obtained from Merck (Darmstadt, Germany). Rapid filtration experiments were done with cellulose nitrate filters (25-mm diameter, 0.2-µm pore size) and a manifold (DN 025/0 Schleicher & Schuell, Dassel, Germany).

Brush-border membrane vesicles were prepared from isolated intestinal epithelial cells in contrast to a whole mucosal homogenate. Epithelial cells were isolated according to the method of Schwenk et al. (24). Briefly, rats were killed by neck fracture. The abdomen was opened, and either a segment of jejunum or distal ileum was removed. Jejunum, defined as the 30 cm distal to the ligament of Treitz, and ileum, defined as the 30 cm proximal to the ileocecal valve, were obtained. The intestine was rinsed and filled with solution A (in mM: 96 NaCl, 27 sodium citrate, 5.6 KH2PO4-K2HPO4, 1.5 KCl). Solution A was then replaced by solution B (in mM: 140 NaCl, 16 KH2PO4-Na2HPO4, 1.5 EDTA, 0.5 dithiothreitol) and palpated gently for 2 min. The intestinal content, containing detached cells, was then drained. Cells and mucus were separated by filtration. The cells were suspended in DMEM at 4°C. Cell viability was >95% as checked regularly by the trypan blue exclusion method.

Preparation of brush-border membrane vesicles was done according to the method of Schmitz et al. (23), as modified by Kessler et al. (13). The following steps were done at 4°C. Cells were centrifuged (2 min, 500 g), and the pellet was suspended in buffer A (in mM: 50 D-mannitol, 12 Tris, pH 7.5). This suspension was homogenized for 30 s at 1,400 rpm (Homogenisator, B. Braun, Melsungen, Germany). Calcium chloride was added to give a final concentration of 10 mM. The suspension was incubated for 20 min before centrifugation at 3,000 g for 15 min (Kontron centrifuge Z 364-K, Berthold, Gosheim, Germany). The supernatant was centrifuged at 27,000 g for 30 min (Beckman L7-55, Munich, Germany). The pellet was suspended in buffer B (in mM: 50 D-mannitol, 10 HEPES, 10 Tris, pH 7.5) and homogenized again. The final pellet was suspended in buffer C (in mM: 0.2 CaCl, 290 D-mannitol, 10 HEPES-KOH, 1 MgCl, pH 7.5) after centrifugation at 27,000 g for 30 min. For anion exchange experiments, buffer C was substituted for buffer D (in mM: 230 D-mannitol, 10 HEPES-Tris, 25 NaHCO3, pH 7.5). Vesicles were visualized with electron microscopy. The vesicles were either used immediately or after short storage at -70°C.

The protein concentration was measured by the Lowry method. Saccharase (6) and leucine aminopeptidase (19) were used as marker enzymes for brush-border membranes. Na+-K+-ATPase was measured spectrophotometrically (22) as a basolateral marker enzyme.

To define the time course of substrate uptake, 60 µl of vesicle suspension were added to 3 ml of the reaction assay at room temperature. Aliquots of 0.5 ml were taken at defined time intervals and transferred immediately to cellulose nitrate filters; the retentate was washed with stop solution (in mM: 130 NaCl, 10 HEPES-Tris, pH 7.5). To measure the initial rate of uptake, 11 µl of vesicle suspension were added to 550 µl of the reaction assay. After 10 s the reaction was stopped by immediate filtration of 500 µl vesicle suspension followed by rinsing with stop solution. Filters were placed in scintillation vials, and scintillation fluid (Quickszint 200, Zinsser Analytic, Frankfurt, Germany) was added. Vials were stored overnight (to eliminate chemiluminescence), and radioactivity was determined using a liquid scintillation counter with automatic external standard for quench correction (Beckman LS 2800).

Data are given as means ± SD. The Student's t-test for unpaired samples was used.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Validation studies. Marker enzymes for brush-border and basolateral poles of the enterocyte were measured to ensure that the isolated brush-border membrane vesicle preparations were of satisfactory purity. Saccharase was enriched seven times (P < 0.001) based on measurement of activity of the brush-border membrane fraction compared with that of the homogenate (Table 1). Leucine aminopeptidase, a second brush-border membrane enzyme, was increased 6.1 times (P < 0.001). Activity of Na+-K+-ATPase, a basolateral marker enzyme, was diminished by a factor of 10 (P < 0.001).

                              
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Table 1.   Activity of marker enzymes for brush-border (saccharase and leucine aminopeptidase) and basolateral (Na+-K+-ATPase) membranes in cell homogenate and after purification of brush-border membrane vesicles

To show that vigorous and reproducible sodium-dependent uptake was present in the brush-border membrane vesicles, glucose was used as a substrate for transport for jejunal brush-border vesicles. For ileal brush-border membrane vesicles, C-tau was used as a substrate.

With the brush-border membrane vesicles from the jejunum, glucose uptake in the presence of sodium ion showed a typical overshoot phenomenon, which was absent when sodium ion was replaced isosmotically by potassium ion (data not shown). With the brush-border membrane vesicles from the ileum, uptake of C-tau in the presence of sodium ion showed a similar overshoot phenomenon. Maximal absorption was reached after 1 min with an uptake of 1.7 ± 0.3 nmol/mg protein. Steady-state uptake was 0.8 ± 0.1 nmol/mg protein (data not shown).

Bile acid transport by jejunal brush-border membranes. The uptake of C-tau was not stimulated by an inwardly directed sodium gradient but trans-stimulated by the presence of an outwardly directed bicarbonate gradient (Fig. 1). By trans-stimulation, absorption rate increased severalfold and showed a typical overshoot phenomenon. Maximum absorption was reached after 1 min with a transport rate of 2.2 ± 0.1 nmol/mg protein. Steady-state levels were 0.6 ± 0.2 nmol/mg protein for the inwardly directed sodium gradient and 0.4 ± 0.2 nmol/mg protein for the outwardly directed bicarbonate gradient.


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Fig. 1.   Uptake of cholyltaurine (C-tau, 100 µM) in jejunal vesicles in presence of inwardly directed sodium gradient [final concentration of buffer inside (in mM): 300 D-mannitol, 10 HEPES-KOH, 1 MgCl2, 0.2 CaCl2; and outside: 100 NaCl, 10 HEPES-Tris, 80 D-mannitol, pH 7.5] or an outwardly directed bicarbonate gradient [final concentration of buffer inside (in mM): 230 D-mannitol, 25 NaHCO3, 10 HEPES-Tris; and outside: 280 D-mannitol, 10 HEPES-Tris, pH 7.5].

In the presence of an outwardly directed bicarbonate gradient, uptake of CDC-tau (Fig. 2) was even higher (5.2 ± 0.7 nmol/mg protein). Steady-state levels for uptake were 1.0 ± 0.6 nmol/mg protein for the inwardly directed sodium gradient and 1.1 ± 0.1 nmol/mg protein for the outwardly directed bicarbonate gradient.


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Fig. 2.   Uptake of chenodeoxycholyltaurine (CDC-tau, 100 µM) in jejunal vesicles in presence of inwardly directed sodium gradient [final concentration of buffer inside (in mM): 300 D-mannitol, 10 HEPES-KOH, 1 MgCl2, 0.2 CaCl2; and outside: 100 NaCl, 10 HEPES-Tris, 80 D-mannitol, pH 7.5] or outwardly directed bicarbonate gradient [final concentration of buffer inside (in mM) 230 D-mannitol, 25 NaHCO3, 10 HEPES-Tris; and outside: 280 D-mannitol, 10 HEPES-Tris, pH 7.5].

Additional experiments were performed to define factors influencing the initial uptake of C-tau during its linear phase. With trans-stimulation with bicarbonate an initial uptake of 110 ± 8 pmol · s-1 · mg protein-1 was observed (Fig. 3). Replacement of bicarbonate by mannitol caused the initial rate of uptake to decrease to 21 ± 7 pmol · s-1 · mg protein-1 (data not shown). Uptake of CDC-tau was more rapid (238 ± 26 pmol · s-1 · mg protein-1) than uptake of C-tau.


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Fig. 3.   Initial rate of uptake of C-tau and CDC-tau (100 µM) by trans-stimulation with bicarbonate in isolated brush-border membrane vesicles of jejunum. Uptake was stopped after 10 s. Effect of coincubation (outside) with CDC-tau, deoxycholyltaurine (DC-tau), bromosulfophthalein (BSP), taurine and SDS (final concentration 250 µM) is shown. Final concentration of buffer (in mM) inside: 230 D-mannitol, 25 NaHCO3, 10 HEPES-Tris; and outside: 280 D-mannitol, 10 HEPES-Tris, pH 7.5. *** P < 0.001.

Uptake showed inhibition when structurally similar bile acids were added to the incubation medium (Fig. 3). Initial rate of uptake of C-tau (100 µM) decreased by 82% to 13 ± 7 pmol · s-1 · mg protein-1 when CDC-tau (250 µM) was added. Similarly uptake of CDC-tau (100 µM) decreased by 77% (55 ± 8 pmol · s-1 · mg protein-1) when DC-tau was added at 250 µM. Addition of bromosulfophthalein, an organic anion with a structure differing from that of bile acids, also decreased the initial rate of uptake of C-tau by 76% to 27 ± 7 pmol · s-1 · mg protein-1. In contrast, addition of taurine (a zwitterionic beta -amino acid) or dodecylsulfate (an amphipathic anion with a hydrocarbon chain skeleton rather than a steroid skeleton) had no effect on the initial rate of uptake of C-tau.

Preloading of the brush-border membrane vesicles with DIDS (a compound known to inhibit anion exchange processes) led to a decrease in the initial rate of uptake by 81% for C-tau (17 ± 8 pmol · s-1 · mg protein-1) and 87% for CDC-tau (32 ± 8 pmol · s-1 · mg protein-1; Fig. 4). Uptake was also temperature sensitive. The performance of the uptake experiment at 0°C decreased the initial rate of uptake of C-tau by 74% to 29 ± 8 pmol · s-1 · mg protein-1. Under the same condition, uptake of CDC-tau decreased by 70% to 71 ± 21 pmol · s-1 · mg protein-1.


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Fig. 4.   Initial rate of uptake of C-tau and CDC-tau (100 µM) by trans-stimulation with bicarbonate in isolated brush-border membrane vesicles of jejunum. Uptake was stopped after 10 s. Effect of preloading vesicles with DIDS or incubation temperature is shown. Final concentration of buffer (in mM) inside: 230 D-mannitol, 25 NaHCO3, 10 HEPES-Tris; and outside: 280 D-mannitol, 10 HEPES-Tris, pH 7.5. *** P < 0.001.

To ensure that the uptake of bile acids occurred into an osmotically active space, uptake was measured as a function of medium osmolarity. Lactulose was used as a nonabsorbable marker. Figure 5 indicates that C-tau uptake was clearly sensitive to an osmotic gradient. Nonetheless, some adsorption of C-tau to the vesicle membrane was observed, presumably because of the known amphipathic properties of the conjugated bile acid anion. A change of the membrane potential by using valinomycin and potassium did not change the uptake of C-tau (data not shown).


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Fig. 5.   Uptake of C-tau (100 µM) in osmotically active space in vesicles under influence of osmotic gradient induced by lactulose.

Plotting the initial rate of uptake against increasing concentrations of substrate (C-tau and CDC-tau) showed a decline in the slope of the transport rate, suggesting a saturable process (Fig. 6). With the Michaelis-Menten equation, kinetics were calculated. The Michaelis constant was 54 µM for C-tau and 29 µM for CDC-tau. The maximal velocity was 180 pmol · s-1 · mg protein-1 for C-tau and 290 pmol · s-1 · mg protein-1 for CDC-tau.


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Fig. 6.   Initial rate of uptake of C-tau and CDC-tau by trans-stimulation with bicarbonate in isolated brush-border membrane vesicles of jejunum. Uptake was stopped after 10 s. Effect of increasing concentration of substrate is shown. Final concentration of buffer (in mM) inside: 230 D-mannitol, 25 NaHCO3, 10 HEPES-Tris; and outside: 280 D-mannitol, 10 HEPES-Tris, pH 7.5. Michaelis constant is 54 µM for C-tau and 29 µM for CDC-tau. Maximal velocity is 180 pmol · s-1 · mg protein-1 for C-tau and 290 pmol · s-1 · mg protein-1 for CDC-tau.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These experiments indicate that two natural taurine-conjugated bile acids that are representative of endogenous bile acids in many vertebrate species are transported across rat jejunal brush-border membrane vesicles by an anion-dependent process. Based on its stimulation by an outwardly directed bicarbonate concentration gradient and its inhibition by DIDS, the process is likely to be an antiport process. Additional lines of evidence that the uptake of conjugated bile acids by these jejunal brush-border membrane vesicles was carrier mediated are 1) saturability, 2) inhibition of uptake by other conjugated bile acids and an organic anion, and 3) temperature dependency of uptake. Transport of CDC-tau was more rapid than C-tau, in contrast to transport by brush-border membrane vesicles from the ileum where the opposite is true. The results thus confirm and extend our previous perfusion studies performed using the guinea pig (2).

Brush-border membrane vesicles were prepared from isolated enterocytes rather than from the total epithelial cell population. The steps required for enterocyte isolation are time consuming, but the final preparation of brush-border membranes was of high purity as evidenced by the enrichment in marker enzymes. Preparation of brush-border vesicles increased the activity of the brush-border marker enzymes by approximately six to seven times compared with the homogenate (Table 1). This increase of marker enzymes of the brush-border membrane is somewhat smaller than in published studies (13), most likely because of the high purity of epithelial cells at the beginning of the vesicle preparation. The marker enzyme for the basolateral membrane, Na+-K+-ATPase, was almost not measurable in the preparation of brush-border membrane vesicles, suggesting that the preparation of brush-border membrane vesicles had negligible contamination by basolateral membranes.

Before the experiments were started, vesicles were kept in a small volume of bicarbonate-rich buffer to preload the vesicles with bicarbonate. At the beginning of the experiment the outside solution was isosmotically diluted (~40 times), leading to a steep outwardly directed concentration gradient of bicarbonate. This dilution step led to a rather high volume of incubation buffer compared with previous studies (13). A drawback of this method is the requirement for a substantial amount of radioactively labeled bile acids.

The nature of the brush-border membrane transport system mediating the uptake of conjugated bile acids in the jejunum is not known. By photoaffinity labeling, an 87-kDa protein that is involved in bile acid transport based on binding of photoaffinity-labeled conjugated bile acids is present throughout the small intestine in the rabbit (15). A similar protein with a molecular mass of 94 kDa is present in the jejunum of the rat (14). The sodium ion-independent organic anion transport protein present in the basolateral membranes of the hepatocyte has been cloned (11, 16) and shown to transport conjugated bile acids. Whether it is present in the brush-border membrane of the jejunal enterocyte has not been reported. It is not present in the proximal and distal colon (11).

The present work indicates that anion exchange transport together with passive absorption of protonated glycine-conjugated bile acids provides an explanation for jejunal uptake of conjugated bile acids in mammals. Such jejunal absorption may not only conserve conjugated bile acids but could modulate intracellular events, as has been claimed for conjugated bile acids in cholangiocytes (1).


    ACKNOWLEDGEMENTS

We thank Dr. Alan F. Hofmann for continuous support.


    FOOTNOTES

This study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft; Am 84/3-1).

Present address and address for reprint requests and other correspondence: A. Amelsberg, Boehringer Ingelheim Pharma KG, Birkendorfer Str. 65, 88397 Biberach, Germany (E-mail: andree.amelsberg{at}bc.boehringer-ingelheim.com).

Received 15 December 1997; accepted in final form 3 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alpini, G., S. Glaser, W. Robertson, J. L. Phinizy, R. E. Rodgers, A. Caligiuri, and G. LeSage. Bile acids stimulate proliferative and secretory events in large, but not small cholangiocytes. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G518-G529, 1997[Abstract/Free Full Text].

2.   Amelsberg, A., C. D. Schteingart, H. T. Ton Nu, and A. F. Hofmann. Carrier-mediated jejunal absorption of conjugated bile acids in the guinea pig. Gastroenterology 110: 1098-1106, 1996[Medline].

3.   Angelin, B., K. Einarsson, and K. Hellstrom. Evidence for the absorption of bile acids in the proximal small intestine of normo- and hyperlipidaemic subjects. Gut 17: 420-425, 1976[Abstract].

4.   Borgstrom, B., G. Lundh, and A. F. Hofmann. The site of absorption of conjugated bile salts in man. Gastroenterology 45: 229-238, 1963.

5.   Christie, D. M., P. A. Dawson, S. Thevananther, and B. L. Shneider. Comparative analysis of the ontogeny of a sodium-dependent bile acid transporter in rat kidney and ileum. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G377-G385, 1996[Abstract/Free Full Text].

6.   Dahlqvist, A. Method for assay of intestinal disaccharidases. Anal. Biochem. 7: 18-25, 1964.

7.   Hagenbuch, B., B. Stieger, M. Foguet, H. Lubbert, and P. J. Meier. Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system. Proc. Natl. Acad. Sci. USA 88: 10629-10633, 1991[Abstract].

8.   Hislop, I. G., A. F. Hofmann, and J. L. Schoenfield. Determinants of the rate and site of bile acid absorption in man (Abstract). J. Clin. Invest. 46: 1070, 1967.

9.   Hofmann, A. F. The enterohepatic circulation of bile acids in health and disease. In: Gastrointestinal Diseases, edited by M. H. Sleisenger, and J. S. Fordtran. Philadelphia, PA: Saunders, 1993, vol. 1, p. 127-150.

10.   Hurwitz, S., A. Bar, M. Katz, D. Sklan, and P. Budowski. Absorption and secretion of fatty acids and bile acids in the intestine of the laying fowl. J. Nutr. 103: 543-547, 1973[Medline].

11.   Jacquemin, E., B. Hagenbuch, B. Stieger, A. W. Wolkoff, and P. J. Meier. Expression cloning of a rat liver Na-independent organic anion transporter. Proc. Natl. Acad. Sci. USA 91: 133-137, 1994[Abstract].

12.   Juste, C., V. Legrand-Defretin, T. Corring, and A. Rerat. Intestinal absorption of bile acids in the pig. Role of distal bowel. Dig. Dis. Sci. 33: 67-73, 1988[Medline].

13.   Kessler, M., O. Acuto, C. Storelli, H. Murer, M. Muller, and G. Semenza. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim. Biophys. Acta 506: 136-154, 1978[Medline].

14.   Kramer, W., G. Burckhardt, F. A. Wilson, and G. Kurz. Bile salt-binding polypeptides in brush-border membrane vesicles from rat small intestine revealed by photoaffinity labeling. J. Biol. Chem. 258: 3623-3627, 1983[Abstract/Free Full Text].

15.   Kramer, W., F. Girbig, U. Gutjahr, S. Kowalewski, K. Jouvenal, G. Muller, D. Tripier, and G. Wess. Intestinal bile acid absorption. Na+-dependent bile acid transport activity in rabbit small intestine correlates with the coexpression of an integral 93-kDa and a periphereal 14-kDa bile acid-binding membrane protein along the duodenum-ileum axis. J. Biol. Chem. 268: 18035-18046, 1993[Abstract/Free Full Text].

16.   Kullak Ublick, G. A., B. Hagenbuch, B. Stieger, C. D. Schteingart, A. F. Hofmann, A. W. Wolkoff, and P. J. Meier. Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver. Gastroenterology 109: 1274-1282, 1995[Medline].

17.   Lewis, M. C., and C. Root. In vivo transport kinetics and distribution of taurocholate by rat ileum and jejunum. Am. J. Physiol. 259 (Gastrointest. Liver Physiol. 22): G233-G238, 1990[Abstract/Free Full Text].

18.   Marcus, S. N., C. D. Schteingart, M. L. Marquez, A. F. Hofmann, Y. Xia, J. H. Steinbach, H. T. Ton-Nu, J. Lillienau, M. A. Angellotti, and A. Schmassmann. Active absorption of conjugated bile acids in vivo. Kinetic parameters and molecular specificity of the ileal transport system in the rat. Gastroenterology 100: 212-221, 1991[Medline].

19.   Martinek, R. G., L. Berger, and D. Broida. Simplified estimation of leucine aminopeptidase (LAP) activity. Clin. Chem. 10: 1087, 1964.

20.   McClintock, C., and Y. F. Shiau. Jejunum is more important than terminal ileum for taurocholate absorption in rats. Am. J. Physiol. 244 (Gastrointest. Liver Physiol. 7): G507-G514, 1983[Abstract/Free Full Text].

21.   Schalm, S. W., N. F. LaRusso, A. F. Hofmann, N. E. Hoffman, G. P. van Berge-Henegouwen, and M. G. Korman. Diurnal serum levels of primary conjugated bile acids. Assessment by specific radioimmunoassays for conjugates of cholic and chenodeoxycholic acid. Gut 19: 1006-1014, 1978[Medline].

22.   Scharschmidt, B. F., B. Keffe, M. M. Blakenship, and R. K. Ockner. Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity. J. Lab. Clin. Med. 93: 790-799, 1979[Medline].

23.   Schmitz, J., H. Preiser, D. Maestracci, B. K. Ghosh, J. J. Cerda, and R. K. Crane. Purification of the human intestinal brush border membrane. Biochim. Biophys. Acta 323: 98-112, 1973[Medline].

24.   Schwenk, M., E. Hegazy, and V. Lopez del Pino. Kinetics of taurocholate uptake by isolated ileal cells of guinea pig. Eur. J. Biochem. 131: 387-391, 1983[Abstract].

25.   Shneider, B. L., P. A. Dawson, D. M. Christie, W. Hardikar, M. H. Wong, and F. J. Suchy. Cloning and molecular characterization of the ontogeny of a rat ileal sodium-dependent bile acid transporter. J. Clin. Invest. 95: 745-754, 1995[Medline].

26.   Sklan, D. Site of digestion and absorption of lipids and bile acids in the rat and turkey. Comp. Biochem. Physiol. A Physiol. 65A: 91-95, 1980.

27.   Sklan, D., P. Budowski, and S. Hurwitz. Site of bile acid absorption in the rat. Lipids 11: 467-471, 1976[Medline].

28.   Spittell, D., L. K. Vongroven, and M. T. Subbiah. Concentration changes of bile acids in sequential segments of pigeon intestine and their relation to bile acid absorption. Biochim. Biophys. Acta 441: 32-37, 1976[Medline].

29.   Stahl, G. E., M. R. Mascarenhas, J. C. Fayer, Y. F. Shiau, and J. B. Watkins. Passive jejunal bile salt absorption alters the enterohepatic circulation in immature rats. Gastroenterology 104: 163-173, 1993[Medline].

30.   Switz, D. M., I. G. Hislop, and A. F. Hofmann. Factors influencing the absorption of bile acids by the human jejunum (Abstract). Gastroenterology 58: 999, 1970.

31.   Wingate, D. L., S. F. Phillips, and A. F. Hofmann. Effect of glycine-conjugated bile acids with and without lecithin on water and glucose absorption in perfused human jejunum. J. Clin. Invest. 52: 1230-1236, 1973[Medline].

32.   Wong, M. H., P. Oelkers, A. L. Craddock, and P. A. Dawson. Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter. J. Biol. Chem. 269: 1340-1347, 1994[Abstract/Free Full Text].

33.   Wong, M. H., P. Oelkers, and P. A. Dawson. Identification of a mutation in the ileal sodium-dependent bile acid transporter gene that abolishes transport activity. J. Biol. Chem. 270: 27228-27234, 1995[Abstract/Free Full Text].


Am J Physiol Gastroint Liver Physiol 276(3):G737-G742
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