on transport conjugated bile salts by intestinal...

Journal of Clinical InvestigationVol. 43, No. 3, 1964

Studies on the Transport and Metabolism of Conjugated BileSalts by Intestinal Mucosa *

MARCR. PLAYOUSTt ANDKURTJ. ISSELBACHER

(From the Department of Medicine, Harvard Medical School, and the Medical Services[Gastrointtestinal Unit], Massachuisetts General Hospital, Bostont, Mass.)

Bile salts have a central role in the digestionand absorption of fat in the intestinal tract. Inaddition, since the sterol nucleus is not furthermetabolized by mammalian cells, the bile saltsrepresent the main excretory products of thebody's cholesterol stores (1). The enterohepaticcirculation of bile salts is well established, andupwards of 959 of the amount excreted throughthe bile duct is absorbed from the intestine forsubsequent re-excretion by the liver (2). Invitro studies have shown an active transportmechanism to be present in the ileum of a numberof species (3, 4); this site of absorption has beenconfirmed in vivo in the guinea pig and the dog(5, 6).

Wehave studied further aspects of the activetransport of bile salts, choosing sodium taurocho-late as a model for most of the work. Wewereunable to find evidence for hydrolysis of theconjugated bile salts during their active trans-port in vitro. Taurocholate absorption acrosseverted gut sacs exhibted saturation kinetics andwas reversibly inhibited by cholate and glyco-cholate. A striking finding was that at low orabsent sodium ion concentrations in the medium,active transport is markedly inhibited. Wehavecompared these effects with those of anoxia andof metabolic inhibitors such as dinitrophenol.

* Submitted for publication September 24, 1963; ac-cepted November 14, 1963.

This work supported in part by U. S. Public HealthService grant A-3014 f rom the National Institutes ofHealth.

t Work done during tenure of a fellowship from thePostgraduate Medical Foundation, University of Sydney.Present address: Department of Medicine, University ofSydney, Australia.

Materials and MethodsCommercial preparations of cholic acid,' glycine,2 and

taurine 3 were recrystallized twice before use. Thepurity of the cholic acid was checked by thin-layerchromatography on silicic acid by the two solvent sys-tems described by Hofmann (7). Solvents employedwere reagent grade and were not redistilled; ether wasperoxide-free. 2,4-Dinitrophenol 3 (DNP) was recrys-tallized from water; other inhibitors were phloridzin 4

and ouabain.5Sodium taurocholate was synthesized by the method

of Norman (8). After evaporation, the reaction mix-ture was dissolved in dilute aqueous alkali and extractedwith ether to remove residual tributylamine. The solu-tion was then acidified and unreacted cholic acid ex-tracted with chloroform. Further purification was car-ried out as described (8) ; however, when there was dif-ficulty with the first recrystallization, a salting out pro-cedure was also employed (9). The purity of the prod-uct was checked by melting point determinations and bythin-layer chromatography; chemical assay for anypersistent contamination by cholate was carried out bythe procedure described below.

Glycocholic acid was synthesized by the technique ofBergstr6m and Norman (10) without modification.The purity of the glycocholate was determined by melt-ing point determinations and thin-layer chromatography.

Radioactive conjugated bile salts were prepared fromnonradioactive cholic acid and either glycine-1-C14 6 ortaurine-S'.7 The S3-taurocholate was essentially radio-chemically pure. On thin-layer chromatography of thesynthesized C14-glycocholic acid, there was a contaminantwith the mobility of glycodeoxycholate; this impuritycould be removed by reverse-phase column chromatog-raphy (11). The absence of unreacted glycine-1-C1' wasdemonstrated by high voltage paper electrophoresis(4,000 v) in formic acid and acetic buffer, pH 1.7 (12).

1 Special enzyme grade. Mann Research Laboratories,Inc., New York, N. Y.

2 Mann Research Laboratories, Inc., New York, N. Y.3 Eastman Organic Chemicals, Rochester, N. Y.4California Corp. for Biochemical Research, Los

Angeles, Calif.5 Eli Lilly and Company, Indianapolis, Ind.6 New England Nuclear Corp., Boston, Mass.7 Nuclear-Chicago, Chicago, Ill.

467

MARCR. PLAYOUSTAND KURT J. ISSELBACHER

Cholic acid and its glycine and taurine conjugates weredetermined colorimetrically by the modified Pettenkofermethod of Irvin, Johnston, and Kopala (13). Other bilesalts are not measured by this method. The bile saltcontent of the wall of an everted gut sac was not usuallymeasured directly but was estimated by subtracting theamounts recovered in the serosal and mucosal com-partments from the quantity added initially to the in-cubation mixture. Taurine was determined by the methodof Pentz, Davenport, Glover, and Smith (14).

Isotopic measurements were made in a Packard liquidscintillation spectrometer. Radioactive substances inaqueous solution were counted in a solution prepared bymixing 6.5 vol of 95%o ethanol with 100 vol of dioxanecontaining 0.7%o 2,5-diphenyloxazole (PPO), 0.01% bis-2-(5-phenyloxazolyl)benzene (POPOP), and 5% naph-thalene. In other cases the radioactive product was dis-solved in 4 ml ethanol and mixed with 10 ml of a tolu-ene counting solution containing 0.3%o PPO and 0.01%POPOP.

Everted gut sac exrperimlents. Female albino rats 8 ofweight 200 g and male golden hamsters 9 weighing 150 gwere fasted overnight before use in these experiments.Each animal was killed by a blow on the head, andeverted sacs were prepared by the method of Wilsonand Wiseman (15). When a sac was to be incubated ina medium of specific electrolyte composition, the gut wasrinsed through with the same solution. Ileal sacs weretaken from the small intestine just proximal to theileo-cecal junction; jejunal sacs were from intestinedistal to the ligament of Treitz. Unless otherwise speci-fied, sacs from rats were 15 cm long and those fromthe hamster 10 cm. In the case of the ileum, more re-producible results were obtained when only one sac wasprepared from each animal.

The solution used on both serosal and mucosal sideswas oxygenated Krebs-Ringer bicarbonate buffer (16)with a glucose concentration of 100 to 200 mg per 100ml and half the usual concentration of calcium. Thecomplete ionic composition of the medium was as fol-lows: Na+, 143; K+, 5.9; Ca'+, 1.27; Mg++, 1.18; Cl-, 125;HC03-, 25; HPO4-, 1.18; and SO4=, 1.18 mM. Sodium-free media were prepared by replacing all the Na+ withK+ or Li+; in experiments with intermediate concentra-tions of sodium, the substitute ion was always K+. Eachsac was filled initially with 2 ml of fluid with the sameionic composition as the mucosal medium; it was thenimmersed in 10 ml of the incubation mixture containingthe bile salt under study, either with or without an in-hibitor. Note that bile salts were not present in theserosal fluid at the start of the incubation. The flaskswere gassed continuously with a mixture of 95% oxy-gen and 5%o carbon dioxide in a Dubnoff shaking incu-bator at 370 C. Under these experimental conditionsthere was no significant fluid transfer from one com-partment to the other. At the completion of the incu-bation each sac was blotted and its contents drained

8 Charles River Laboratories, Boston, Mass.9 Dennen Animal Industries, Inc., Gloucester, Mass.

into a tared vessel that was then reweighed; a sample ofmucosal fluid was also taken for analysis. Protein wasprecipitated by the addition of 0.1 ml 80% trichloroaceticacid or by boiling.

Mucosal homogenates. After removal of the intes-tine, the epithelial cells were gently scraped free as de-scribed previously (17), and 7 ml of a solution of 0.125M potassium phosphate buffer (pH 7.0) was added foreach gram of cells. The homogenates were preparedwith a Potter-Elvehjem tissue grinder with a Teflonpestle. The homogenate was subsequently filteredthrough a double layer of absorbent gauze. The stand-ard incubation mixture contained 0.5 ml of the mucosalhomogenate, 4 mg glucose, 800 mAimoles sodium tauro-cholate, and 1.5 ml 0.125 M potassium phosphate buf-fer, pH 7.0. Incubations were carried out for 90 min-utes at 370 C in air with occasional agitation and wereterminated by immersion in boiling water for 10 minutes.

Analysis of taurocholate and cholate mixtulres. Whenit was necessary to determine in the same specimen theconcentrations both of taurocholate and of cholate, thefollowing procedure was adopted. The sample was acidi-fied and extracted three times with 2 vol of ether; thepooled ether extract was washed with a small volumeof 0.1 N hydrochloric acid that was subsequently addedto the water phase. After neutralization and evapora-tion at 400 C under a stream of air, an appropriate frac-tion of the residue was analyzed by the colorimetricmethod of Irvin and associates (13). Standard solu-tions were extracted in an identical way during eachset of estimations. The recovery of taurocholate in thewater phase was almost complete; that of cholate in theether extract was approximately 85 to 90%.

Estinmation of specific activity of transported S35-taiuro-cholate. After protein precipitation with trichloroaceticacid, a portion of the supernatant liquid was extractedfour times with ether (saturated with 0.1 N HCl). Ap-proximately 5.5 mg nonradioactive taurine was added tothe aqueous phase, which was then evaporated at 400 Cunder a stream of air. The residue was taken up in 5ml 98%o ethanol, and, after centrifugation, the super-natant fluid was assayed for radioactivity and the con-centration of taurocholate determined chemically.

Estimation of spccific activity of trantsportcd C14-gly-cinie-labeled glycocholate. Protein was precipitated byimmersion of the specimen for 10 minutes in boiling wa-ter. The supernatant fluid was decanted and acidified;after addition of excess nonradioactive glycine, a portionof the supernatant liquid was extracted with ether. Theether fraction was subsequently evaporated and the resi-due redissolved in absolute ethanol. The glycocholicacid (dissolved in the supernatant fluid) was then as-sayed for radioactivity and its concentration determinedchemically; thin-layer chromatography did not demon-strate any contamination with cholic or taurocholic acids.

Analysis for C14-glycine. In experiments designed todetermine if there was hydrolysis of glycine-labeled gly-cocholate during its transport through an everted sac,it was necessary to assay the serosal fluid for C04-glycineafter removal of all radioactive glycocholate. After

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TRANSPORTAND METABOLISMOF BILE SALTS

TABLE I

Transport of sodium taurocholate by everted rat intestinal sacs*

Serosal bile salt concentrationMucosal taurocholate Mucosal taurocholate after incubationconcentration before concentration after

Region of intestine incubation incubation Taurocholate Cholate

mpmoles per mlJejunum 0 42 36 6

200 240 86 0200 204 86 0

1,000 986 122 41,000 920 148 4

Ileum 0 20 58 8200 50 386 20200 50 478 8

1,000 660 1,050 21,000 680 476 14

* The mucosal medium in each experiment was 10 ml Krebs-Ringer bicarbonate buffer containing 10 mg glucoseand taurocholate at the concentrations indicated. The sacs were 15 cm in length and were filled with 2 ml Krebs-Ringerbicarbonate solution containing 2 mgglucose but no taurocholate. Incubations were carried out for 90 minutes at 370 C,and the flasks were gassed continuously with 95% oxygen and 5%carbon dioxide.

precipitation of protein by heating to 1000 C for 10 min-utes, carrier glycine was added, and most of the bile saltwas extracted into ether. The remainder of the glyco-cholate was removed by extraction into butanol, and theglycine was left in aqueous solution. Controlled experi-ments showed that the separation of glycine and glyco-cholate was virtually complete and that the glycine re-

covery exceeded 95%; high voltage electrophoresis con-

firmed the absence of radioactive glycine contaminant inthe glycocholate fraction.

Results

Experiments to determine if conjugated bile saltsare hydrolyzed during transport

1) Taurocholate. Everted intestinal sacs were

incubated with sodium taurocholate and the serosalfluid analyzed chemically for cholate and fortaurocholate. The results in Table I show activetrasport of taurocholate by the rat ileum; in thetwo experiments with the lower bile salt concen-

tration, the final ratios between serosal and mu-

cosal concentrations (S/M ratios) were 7.7 and9.6, respectively. No significant amounts of cho-late were demonstrated in the serosal fluid, andresults with sacs prepared from hamster ileumwere similar. Sacs prepared from the jejunum didnot demonstrate active transport of taurocholate.These findings concerning the site of bile salttransport are consistent with previous work (3).

The possibility remained that taurocholate was

split and then reconjugated during its passage

through the wall of the everted sac. This was

investigated by determining the specific activity

of S35-taurocholate transported in the presenceand absence of nonradioactive taurine. In otherexperiments we observed that part of the taurinewas transported during these experimental con-ditions and that these concentrations of taurinedid not inhibit the transport of cholate or tauro-cholate. The absence of an alteration in specificactivity excludes significant hydrolysis and re-

TABLE II

Specific activity of S35-taurocholate transported byeverted ileal sacs in the presence of taurine*

Specific activityof transported

Initial S35-taurocholatetmucosaltaurine No. of SE or

Animal concentration observations Mean range

pmoles per mlRat 0 10 75 +3.9t

0.1 8 75 +3.51.0 9 73 :+:4.0

Hamster 0 4 86 79-93 §0.2 3 88 86-911.0 3 84 82-864.0 4 88 84-93

* The mucosal medium in each experiment was 10 mlKrebs-Ringer bicarbonate buffer containing 2 ,umoles335-taurocholate, 10 mgglucose, and taurine in the concen-trations indicated. Rat sacs were 15 cm in length; hamstersacs were 10 cm. Each sac was filled with 2 ml Krebs-Ringer bicarbonate buffer containing 2 mg glucose butneither taurocholate nor taurine. Incubations were for90 minutes at 370 C.

t Specific activity of S35-taurocholate in the serosal fluidafter incubation is expressed as percentage of the specificactivity of the substrate added initiallv to mucosal side.

± Standard error of the mean.§ Range.

469

MARCR. PLAYOUSTANDKURTJ. ISSELBACHER

synthesis of taurocholate (Table II). The con-sistently lower specific activity in the rat experi-ments is presumably due to this animal's lack ofa gall bladder and the resultant greater dilutionwith endogenous nonradioactive taurocholate.

Homogenates prepared from rat and hamsterintestinal mucosa were incubated with sodiumtaurocholate (0.4 umole per ml) for 90 minutesand were then analyzed for cholate and tauro-cholate. No significant splitting of the conjugatedbile salt was observed; the results of a typical ex-periment are given in Table III. In other ex-periments rat jejunal and ileal homogenates wereincubated with a greater concentration of sodiumtaurocholate (10 ,umoles per ml), and demon-stration of the liberation of free taurine was im-possible. The accuracy of the taurine estimationwas such that any hydrolysis in excess of 2%would have been detected.

2) Glycocholate. Everted ileal sacs from ratand hamster were incubated in Krebs-Ringer bi-carbonate buffer containing C14-glycocholate (gly-cine-labeled) at a concentration of 0.43 /Amole perml and nonradioactive glycine at a concentrationof 2.67 ,umoles per ml. Each sac was filled with2 ml of buffer without labeled bile salt or gly-cine. After an incubation of 45 minutes the sero-sal fluid was analyzed for C14-glycine and C14-glycocholate. All the radioactivity in the serosalfluid was present in the conjugated bile salt frac-tion and none in free glycine. The sac walls werethen homogenized and analyzed with the same

TABLE III

Incubation of mucosal homogenateswith sodium taurocholate*

Bile salt recoveredafter incubation

Description ofAnimal homogenate Taurocholate Cholate

,umolesRat Ileal, heat inactivatedt 890 2

Ileal 904 6Jejunal 800 0

Hamster Ileal, heat inactivatedt 770 0Ileal 776 12Jejunal 814 0

* The incubation medium consisted of 0.5 ml homo-genate, 800 msmoles sodium taurocholate, 4 mg glucose,and 1.5 ml 0.125 Mpotassium phosphate buffer, pH 7.0.The incubation was at 370 C for 90 minutes.

t Homogenate prepared from ileal mucosa was in-activated by immersion in boiling water for 15 minutes.

result. The final S/M ratio for the bile salt was9.5 in the rat sac and 3.9 in the hamster sac.Parallel sac experiments were conducted with la-beled glycine and nonradioactive glycocholateat the same concentrations. Glycine passed fromthe mucosal to the serosal side, but under theseexperimental conditions a concentration gradientwas not established; the final S/M ratios were0.39 in the rat and 0.83 in the hamster. Therewas no exchange of label between the C'4-glycineand the glycocholate.

In other experiments with everted sacs pre-pared from hamster ileum the specific activity oftransported C14-glycocholate (glycine-labeled) wasdetermined and was found to be unaltered duringthe simultaneous transport of nonradioactive gly-cine. These data excluded the possibility thathydrolysis of glycocholate occurred at one stageof transport followed later by resynthesis of theconjugated bile salt.

Transport of cholate

Sacs from hamster ileum were incubated for 45minutes in a medium containing sodium cholatein a concentration of 0.18 pmole per ml (therewas no bile salt initially on the serosal side).Cholate accumulated in the serosal compartment,the final S/M ratio being approximately 2. Thisconcentration gradient is considerably lower thanthose found for taurocholate and glycocholate un-der similar conditions.

Wewished to determine whether the intestinalmucosa was able to synthesize taurocholate whenboth cholate and taurine were present together.Everted hamster ileal sacs were incubated for 1hour in media containing sodium cholate (0.5,umole per ml) and taurine (1.0 ,umole per ml).At the end of the incubation the serosal fluid wasanalyzed; the mean cholate concentration was 0.2Mumole per ml, and in each case there was only atrace of taurocholate in a concentration that didnot exceed the levels found in control sacs in-cubated without added bile salts and taurine.

Effect of inhibitors and of electrolytes on tauro-cholate transport

Taurocholate transport by sacs of everted ham-ster ileum was studied in the absence of oxygen andin the presence of DNPand of ouabain. The re-

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TABLE IV

Inhibition of taurocholate transport acrosseverted hamster ileal sacs*

Final taurocholateconcentration

in serosal fluid

Incubation conditions No. of experiments Mean SE

mjsmoles per mlControl 9 498 ±34Anoxia, 95%N,5O%C02 5 71 i8DNP, 2.5 X 10-3 Mt 5 215 47Ouabain, 3.4 X 10- M 5 485 420Na+-free

(replaced by K+) 5 16 ±t2Na+-free

(replaced by Li+) 5 55 ±7

* The usual mucosal medium was 10 ml Krebs-Ringerbicarbonate buffer containing 2 jumoles sodium tauro-cholate and 10 mgglucose. The sacs were 10 cm in lengthand were filled with 2 ml Krebs-Ringer bicarbonate buffercontaining 2 mg glucose but neither taurocholate norinhibitors. In the Na+-free experiments, Na+ in both themucosal medium and the serosal fluid was replaced byequimolar concentrations of either K+ or Li+. Incubationswere at 370 for 45 minutes, and the flasks were gassed con-tinuously with 95% oxygen and 5%carbon dioxide exceptas indicated above.

t DNP= 2,4-dinitrophenol.

sults in Table IV indicate that both anoxia andDNPat a concentration of 2.5 x 10-13 M causedsignificant inhibition of taurocholate transportinto the serosal medium. The amounts of bilesalt retained in the sac walls were reduced pro-

portionately. Results of transport in the presence

1.0

s0.8 Serosal -medium

iitSac Wall~j0.6

0.4

0

145 120 100 80 60 40 20 0

No" CONC. lIN MEDIUIM mM

FIG. 1. EFFECT OF NA+ CONCENTRATION ON TAURO-

CHOLATETRANSPORTBY EVERTED SACS OF HAMSTERILEUM.

The incubation conditions were the same as described

in Table IV. Variation of the Na" concentration in

mucosal and- serosal media was achieved by replace-

ment of Na" by equimolar concentrations of K+. Re-

sults at each Na+ concentration level are means of 5

to 10 experiments; standard errors of the means are

indicated by vertical lines.

of ouabain at a concentration of 3.4 x 10-5 Mdidnot differ significantly from control values (Ta-ble IV).

The omission of glucose from the incubationmedia or the addition of phloridzin 10-5 M (a con-centration that inhibits active transport of glu-cose) had no effect on taurocholate transport.

Striking results were noted when the electrolytecomposition of the medium was altered. In a so-dium-free medium, where the Na+ was replacedby K+, practically no taurocholate reached theserosal side. When Li+ replaced Na+, the finalserosal concentration of bile salt was slightlygreater (Table IV). The absence of calcium andmagnesium, either individually or together, didnot affect taurocholate transport.

The dramatic influence of sodium ions wasfurther studied in a series of experiments in mediawith varying sodium concentrations and in whichthe replacing ion was potassium. Figure 1 showsthat as the Na+ concentration was reduced, therewas a progressive decrease in the serosal accumu-lation of taurocholate; there was, however, no in-

CHOLATE 1.95 mM oKi -1.7 mM / CHOLATE0.98mM

Ki = 1.6mM

1/v2-

No Inhibitor

0

0

0 5 t0I/s (mm),

FIG. 2. TRANSPORTOF S'-TAUROCHOLATE BY HAMSTERILEAL SACS IN PRESENCEAND ABSENCE OF SODIUM CHO-LATE (Lineweaver-Burk plots). Incubation conditionswere the same as described in Table IV except that thetaurocholate concentrations in the mucosal media rangedfrom 0.1 to 7.4 mM. 1/S is the reciprocal of the ini-tial taurocholate concentration. 1/V is the reciprocalof the.amount of S'-taurocholate disappearing from themucosal medium. The lowermost line is for uninhibitedtaurocholate transport; each point is the mean of 2 to 8experiments. The upper two lines are for taurocholatetransport in the presence of sodium cholate; each pointis the result of one experiment.

471


hibition of bile salt uptake into the sac wall untilthe Na+ concentration was below 80 mM.

Kinetic studies of bile salt transport

Transport of S35-taurocholate was studied inhamster ileal sacs, the initial bile salt concentra-tion on the mucosal side ranging from 0.1 to 7.4mM. Absorption was measured by the disap-pearance of radioactivity from the mucosal com-partment over a 45-minute incubation and wasplotted in reciprocal form against the reciprocalof the original substrate concentration [Line-weaver and Burk plot (18)]. The results fall ona straight line, suggesting that absorption of tauro-cholate conforms to the pattern of saturationkinetics described by Michaelis and Menten (19).The regression line for results from 46 experimentswas calculated by the least squares method, andthe apparent Km (half maximal concentration)was found to be 1.34 mM (Figure 2) 'and theVmax (apparent limiting velocity of absorption),10.9 MAmoles for a 10-cm sac incubated 45 minutes.By the same method the Km values for sodiumcholate and for sodium glycocholate were 1.95 mMand 0.90 mM, respectively. The use of initialsubstrate concentration uncorrected for the fall inconcentration during absorption is common prac-tice and has been justified mathematically byFisher and Parsons (20); however, the calcu-

GLYCOCHOLATE I.72 mM 0 GLYCOCHOLATE0.86 mMKi - 1.0 ml * Ki 1.1mM

3-0

1/ v

0 5 10/ S (m M)1I

FIG. 3. TRANSPORTOF S'5-TAUROCHOLATE BY HAMSTER

ILEAL SACS IN PRESENCEAND ABSENCEOF SODIUM GLYCO-CHOLATE (Lineweaver-Burk plots). The experimentalconditions were the same as described in Figure 2 ex-

cept that the inhibiting bile salt was sodium glycocholate.

lated value for the Kmis different if the correc-tion is made.

When sodium cholate or sodium glycocholatewas in the mucosal medium together with S35-taurocholate, there was definite inhibition oftaurocholate absorption by the everted sacs.Double reciprocal plots were drawn for taurocho-late absorption in the presence of each of the twobile salts at two concentration levels (Figures 2and 3). These values evidently tend to fall onstraight lines which, when extrapolated, intersectthe y-axis at or close to the poiInt where it iscrossed by the regression liine for uninhibitedtaurocholate transport.

These results, therefore, are consistent withcompetitive rather than noncompetitive inhibition.The Ki values for cholate and glycocholate (eachat two concentration levels) were calculated fromthe experimental data by assuming that they arecompetitive inhibitors of taurocholate transportand that the Km for taurocholate is 1.34 mM(Figures 2 and 3).

Discussion

Studies on the enterohepatic circulation of bilesalts have indicated that their reabsorption fromthe intestine is very efficient. For example, in anormal 200-g rat, the bile acid pool of 20 mg hasbeen calculated to circulate about tenl times eachday; only about 5 mg remains unabsorbed and isexcreted in the feces after modification by the bac-teria of the large intestine (2, 21).

The bile acids, as excreted by the liver, are con-jugated with glycine or taurine, and in most spe-cies these compounds remain unhydrolyzed whilewithin the lumen of the small intestine (22).The conjugated bile salts are water soluble buthave a molecular size too large to be absorbed bydiffusion through membrane pores (23). ThepH of intestinal contents is such that the bile saltsare for all practical purposes fully ionized, thusmaking unlikely any significant absorption by amechanism involving the solution of the undis-sociated acid in the lipid components of the mu-cosal cell membrane [the pKa of taurocholic acidis 1.54 and of glycocholic acid 4.54 (24)]. Wechose sodium taurocholate for particular studybecause the limited lipid solubility and low pKa ofthe free acid suggested that its absorption wouldbe more dependent on a specific transport mecha-

472


nism. With regard to our in vitro data, wemight note that the pH gradient across evertedileal sacs from the hamster would favor accumu-lation of a weak acid on the mucosal and not onthe serosal side (25).

It has been recognized recently that the ab-sorption of many compounds is accompanied bytheir metabolic modification at the surface of themucosal cell or within the cell. For example,hydrolysis or esterification is important in the ab-sorption of disaccharides (26), peptides (27), andlipids (28). In everted gut sacs some steroidhormones and thyroxine analogues have beenshown to appear on the serosal side as conjugatesof glucuronic acid (29-31). A reverse type ofmechanism exists in the case of the water solublebilirubin diglucuronide, which appears not to beabsorbed as such but only after hydrolysis to thelipid soluble free bilirubin (32, 33). There issome evidence in rats to suggest that this hydroly-sis takes place at the mucosal cell surface ratherthan in the intestinal lumen (32).

Active transport of conjugated bile salts haspreviously been demonstrated in the ileum (3),although there have been no data on the pos-sibility of hydrolysis during absorption. Ineverted ileal sacs from the rat and the hamster,we have confirmed the presence of a mechanismfor the transport of taurocholate, glycocholate,and cholate against a concentration gradient; thisproperty was absent in jejunal sacs. We wereunable to demonstrate the hydrolysis of eithertaurocholate or glycocholate during its transport.Likewise the possibility of hydrolysis followed byreconjugation has been excluded. Confirmatoryexperiments with mucosal homogenates failed todemonstrate a hydrolytic mechanism (peptidase)for the conjugated bile salts. Therefore, the ac-tive transport of the conjugated bile salts stud-ied clearly does not depend on their metabolicmodification by hydrolysis.

Absence of sodium ions has been shown to in-hibit the intestinal active transport of monosac-charides (34-6), amino acids (37, 38), pyrimi-dines (37), and more recently inorganic phos-phate ions (38) ; indeed the effect is not limitedto the intestine but has been demonstrated inother tissues such as the kidney (39). Studies ofsugar transport into strips of hamster intestinehave led Crane, Bihler, and Hawkins to postulate

that glucose enters the mucosal cell as a Na+-glucose-carrier complex which then dissociatesinside the cell, the Na+ being removed immediatelyfrom the cell interior by a Na+ pump (40, 41).They suggested that the inhibitory effects ofcardiac glycosides, anoxia, and dinitrocresol aredue to a failure of the Na+ pump, such that al-though glucose could still enter and leave the mu-cosal cell freely (coupled with Na+ and carrier),no uphill gradient could be established.

In our experiments with taurocholate, the re-sult of replacing Na+ in the medium by K+ wasstriking. Under these conditions virtually notaurocholate appeared in the sac wall or in theserosal compartment. The use of Li+ as the re-placing ion was associated with somewhat lessinhibition; a similar difference between the ef-fects of K+ and Li+ media was noted by Bihler andCrane in the case of sugar transport (35). Whenthe incubations were carried out with Na+ butunder anoxic conditions or in the presence ofDNP, taurocholate entered the sac wall andpassed to the mucosal side, but it was not con-centrated. Ouabain, which in low concentration isconsidered to be primarily an inhibitor of Na+transport (42), has been shown to depress thetransfer of sugars and of amino acids (43, 44),but we did not find a significant effect on tauro-cholate transport. In accord with the conclusionsof Crane in regard to glucose transport, our datasuggest that sodium ions are necessary for thenonenergy dependent entry of taurocholate intomucosal cells, whereas the subsequent intracel-lular and serosal accumulation against a concen-tration gradient requires both Na+ and energy.

When we used incubation media with graduallydiminishing Na+ concentration, there was a pro-gressive decrease in the serosal accumulation oftaurocholate (Figure 1). There was no inhibitionof taurocholate uptake in the sac wall until theNa+ concentration fell below 80 mM. Althoughassessing the significance of this finding is diffi-cult at present, it is possible that sodium depriva-tion acts at two points: 1) at the site of entry ofthe bile salt into the mucosal cells and 2) at somepoint in the transport of taurocholate from themucosa to the serosal compartment. Thus, atmoderate reductions of Na+ concentration, theconcentration gradient achieved between the tis-sues of the sac wall and the mucosal medium is

473


virtually normal, but there is a significant inhibi-tion of taurocholate transport into the serosalfluid.

Sodium ions are themselves actively trans-ported by the small intestine, and the normiial po-tential difference between mucosal and serosalsurfaces can be explained on this basis (45).The membrane charge is diminished in the pres-ence of low mucosal Na+ concentration or in theabsence of a sugar that can be actively transported(46). The magnitude of the electrical gradientis lowest in the ileum (45), and whether it canplay a significant role in assisting taurocholatetransport is doubtful. However, the transport ofthe large bile salt anion with Na+ as an ion pairis quite possible.

Since the active transport of a large number ofsubstances, mostly nonelectrolytes, is dependenton the presence of Na+, the association is prob-ably a fundamental one (47). A promising ap-proach is suggested by the Mg++ dependenit (Na++ K+-activated) membrane, adenosine triphos-phatase (ATPase), which was first demonstratedby Skou in crab nerve (48). This enzyme hasbeen found in a number of tissues (49) and inexperiments on unfragmented red cell ghosts hasbeen shown to be part of a coupled transport sys-tem for Na+ and K+, activated inside the cells byNa+ and outside by K+ (50). One can speculatethat in the intestine a Na+-activated membrane,ATPase, may provide the metabolic energy for anumber of different carrier-mediated transportsystems including the one for taurocholate.

That the transport of taurocholate, cholate, andglycocholate obeyed Michaelis-Menten kinetics isconsistent with the hypothesis that the rate-limitingfactor in the absorption of each compound is itscombination with a membrane carrier molecule.Cholate and glycocholate were found to cause re-versible inhibition of taurocholate transport, andtheir Ki values at two concentration levels corre-sponded reasonably well with their respective Kmdeterminations when they were transported alone.These data suggest that the three bile salts sharethe same active transport pathway. Although thistype of evidence has been widely used to specifythe absorption characteristics of many compounds,it must be recognized that the assumptions in-volved in the derivation of the Michaelis-NiMentenequation are valid only in relatively simiiple purified

enzyme systems, and one must hesitate beforeconcluding that these same assumptions also applyin the much more complex process of intestinalabsorption (51, 52). Furthermore, transport bymechanisms unrelated to membrane carriers canin some circumstances show saturation and com-petition kinetics (53).

Lack and Weiner have already shown mutualinhibition of transport by a number of bile acids(4), but interpretation is difficult because many ofthese compounds, particularly the unnconjugatedmono- and dihydroxy bile acids are toxic to theintestinal mucosa and depress absorption generally(4).

Further information on the structural speci-ficity of the transport system for bile salts is im-portant because they are the major end products ofcholesterol metabolism. Inhibition of their reab-sorption from the intestine might therefore beexpected to result in increased conversion of cho-lesterol to bile salts and a diminution of the cho-lesterol pool. In somne species of animals and inman, hypocholesterolemia has been shown to fol-low the oral administration of an intestinal bilesalt sequestrant such as cholestyramine (54), butsteatorrhea is a frequent complication. It is theo-retically possible that a structural analogue of thebile salts may cause a specific block in their ilealreabsorption yet not interfere with their functionin fat digestion and absorption.

Summary1. The transport of taurocholate, glycocholate,

and cholate has been studied with everted intestinalsacs from the hamster and the rat. The pres-ence in the ileum of an active transport mechanism(transport against a concentration gradient), andits absence in the jejunum, have been confirmed.

2. No evidence was found for hydrolysis of theconjugated bile salts during the process of ab-sorption, nor were they hydrolyzed after incuba-tion with homogenates of intestinal miiucosa. Con-versely, when cholate transport was studied in thepresence of excess taurine, there was no evidenceof taurocholate synthesis by the mucosa.

3. In the absence of Na+ in the mucosal medium,taurocholate transport was markedly inhibited, andthis inhibition was greater than that caused byanoxia or by dinitrophenol. An apparent dis-sociation was noted between the effects of Na+

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deprivation on taurocholate accumulation in thesac wall and in the serosal fluid medium. Therelationship of Na+ and bile salt transport wasdiscussed. The participation of a Na+-activatedmembrane adenosine triphosphatase may be op-erative in the taurocholate active transport mecha-nism.

4. Transport of taurocholate, cholate, and gly-cocholate was shown to obey Michaelis-Menten(saturation) kinetics, and there was evidence thatcholate and glycocholate are competitive inhibitorsof taurocholate transport. Wesuggest that tauro-cholate, glycocholate, and cholate share the sametransport mechanism.

Acknowledgments

The authors are indebted to Miss Dorothy Budz andto Mrs. Marilyn Kozacko for valuable technical assistance.

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