Active absorption of conjugated bile acids in vivo

GASTROENTEROLOGY

1991;100:212-221

Active Absorption of Conjugated Bile Acids In Vivo Kinetic Parameters and Molecular Specificity of the Ileal Transport System in the Rat SAMUEL N. MARCUS, CLAUDIO D. SCHTEINGART, MARGARET L. MARQUEZ, ALAN F. HOFMANN, YUN XIA, JOSEPH H. STEINBACH, HUONG-THU TON-NU, JAN LILLIENAU, M. ANTONIETTA ANGELLOTTI, and ADRIAN SCHMASSMANN Division of Gastroenterology, La Jolla, California

Department

of Medicine, University

Active transport of conjugated bile acids by the distal ileum is required for efficient enterohepatic cycling of bile acids. Experiments were performed in the rat to obtain accurate values for T,,,, and Michaelis constant (K,,,)of the absorptive area of the rat ileum and to define the structural specificity of the transport system. The distal fifth (20 cm) of the small intestine from an anesthetized animal with a biliary fistula was perfused using solutions of 10 taurine-conjugated bile acids; a flow rate was used that was sufficiently high such that unstirred water layer effects were negligible and the intraluminal concentration remained unchanged throughout the perfused segment. The absorption rate was equated with the rate of hepatic bile acid secretion. Values of T,, (kmol/min * kg) were markedly influenced by bile acid structure: cholyltaurine, 12.9; ursocholyltaurine, 9.6; ursodeoxycholyl taurine, 5.0; and lagodeoxycholyl-(3cY,12@dihydroxy-cholanoic acid)taurine, 1.2. Decreasing the length of the side chain of ursodeoxycholate conjugates from 6 to 6 carbon atoms was associated with a modest increase in T,, values from 5.0 to 9.1 kmol/min . kg. Values of K,,, correlated with T,, values and ranged from 0.5 to 5 mmol/L, being highest for those bile acids that were best transported. The T,, for cholyltaurine transport was not reached when the intraluminal concentration was as high as its critical micellization concentration, precluding the definition of its T,,,; however, for ursocholyltaurine, with a critical micellization concentration of 40 mmol/L, saturation of transport was clearly shown. Kinetic parameters could not be obtained for two common dihydroxy conjugates (chenodeoxycholyltaurine and deoxy-

of California at San Diego,

cholyltaurine) because at a transport rate of 2 pmol/ min * kg systemic toxicity and death occurred. These studies define the maximal transport capacity of the rat ileum for taurine-conjugated bile acids; they indicate that the ileal transport system in the rat is of low affinity and high capacity for taurine conjugates of hydrophilic bile acids, and they show that both nuclear substituents and side chain length influence the transport rate of taurine-conjugated bile acids.

he presence of conjugated bile acids in the small intestinal lumen at an aqueous concentration above their critical micellization concentration (CMC) is necessary for the efficient absorption of dietary lipids (1). Achievement of a high aqueous concentration of bile acids in the small intestine requires high bile acid secretion rates, which can only occur because of the accumulation of a large circulating pool of bile acids (2). This accumulation, in turn, results from an active transport process located in the distal ileum (3). Loss of ileal conservation of bile acids has a

T

Abbreviations used in this paper: CDCA, chenodeoxycholic acid; CDC-tau, chenodeoxycholyltaurine; CMC, critical micellization concentration; C-tau, cholyltaurine; DCA, deoxycholic acid: DC-tau, deoxycholyltaurine; HPLC, high-pressure liquid chromatography; iso-UDCA, iso-ursodeoxycholic acid; iso-UDC-tau, isoursodeoxycholyltaurine; LDCA, lagodeoxycholic acid: LDC-tau, lagodeoxycholyltaurine; nor-UC-tau, nor-ursocholyltaurine; norUDCA, nor-ursodeoxycholic acid; nor-UDC-ams, nor-ursodeoxy cholyl-nor-taurine; nor-UDC-tau, nor-ursodeoxycholyltaurine; PE, polyethylene; RRT, relative retention time; UC-tau, ursocholyltaurine; UDC, ursodeoxycholate; UDC-tau, ursodeoxycholyltaurine. 8 1991by the American Gastroenterological Association 0016-5085/91/$3.00

January 1991

number of clinical consequences such as secretory diarrhea or decreased bile acid secretion causing fat maldigestion and malabsorption (4,5). Because conjugated bile acids are efficiently absorbed from the ileum and because, at least in humans, de novo bile acid biosynthesis from cholesterol makes up < 5% of bile acid secretion (6) the flux of bile acids through the ileum must be nearly equal to that through the liver. Published figures for rates of bile acid secretion during active enterohepatic cycling in the rat average 2-6 tJ,mol/min . kg (7-ll),figures that seem considerably higher than published values for the T,,, of conjugated bile acid transport by the distal rat intestine (12). Several previous studies (e.g., 12, 13) have used in vitro sac preparations and have equated loss from the luminal solution with absorption. Perfusion studies of absorption have also been performed (14,15), but in these experiments unstirred layers were not considered and saturation of transport was not always obtained. It is now recognized that active bile acid absorption by the ileal enterocyte involves at least two carrier systems: the first is a sodium-coupled cotransport system located in the apical membrane (16-18); the second is an anion-exchange system located in the basolateral membrane (18,19). These considerations suggested that additional information was needed on the kinetics of ileal transport in the rat, and such studies are reported here. It was judged important to characterize the kinetics of transcellular flux under steady-state conditions in which the concentration during absorption was known and diffusion through the unstirred layer was not rate-limiting. It was also thought important to define total ileal transport capacity by perfusing an ileal segment of sufficient length to include most of the area where active ileal absorption occurs. Because the capacity of the jejunum or colon for active transport of conjugated bile acids is quite low, the value obtained would be slightly less than the T,,, for the entire intestine, i.e., the entire animal (12,14,15). It seemed desirable to study the transport of a variety of bile acids varying widely in hydrophobicity and CMC values. Taurine-conjugated bile acids were used because such compounds are strong acids (pK,, < 1)and fully ionized at ileal pH; passive absorption of such compounds should occur only to a very limited extent, and, accordingly, most absorption should be active. Finally, because the aggregation of bile acid molecules to form micelles has complicated the interpretation of previous transport studies, the transport of conjugated bile acids varying widely in CMC values were examined over a wide range of concentrations to test whether saturation of transport could be shown to occur at concentrations well below the CMC, i.e., when bile acids were present solely in monomeric

ABSORPTION OF CONJtJGATED BILE ACIDS IN VIVO

213

form. The results reported here resolve the previous discrepancies in the literature and also provide new information on the ileal transport system for bile acids in the rat. Materials

and Methods

The chemical structure, trivial names, tions of the compounds studied are shown in z gives their relative retention times (KRT) by liquid chromatography (HPLC) (20) and CMC mol/L Na’ (21).

and abbreviaTable 1. Table high-pressure values at 0.15

Bile Acids Unconjugated bile acids. Deoxycholic acid was purchased from Aldrich Chemical Co. (Milwaukee, WI). Lagodeoxycholic acid (LDCA; 3a,l2(3-dihydroxy-5B-cholan-24-oic acid) (22), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), and nor-ursodeoxycholic acid (nor-UDCA) were gifts from Diamalt AG (Raubling, Germany). Ursocholic acid was a gift from Gipharmex (Milan, Italy). Conjugated bile acids. Cholyltaurine (taurocholate) was purchased from Aldrich and purified by adsorptioncolumn chromatography with chloroform-methanol mixtures used as eluant. Iso-ursodeoxycholic acid (iso-UDCA) was synthesized in this laboratory as described (23). Other bile acids were conjugated by EEDQ-catalyzed coupling with taurine or N-methyl taurine as described by Tserng et al. (24). Conjugates were purified by column chromatography using silica gel and chloroform-methanol mixtures and were at least 98% pure by HPLC (21) and by thin-layer adsorption chromatography using a solvent system for conjugated bile acids (25). Nor-ursocholyltaurine (nor-UCtau) was a gift from Professor Bengt Borgstrom (Lund, Sweden]. Details of the synthesis have been published (26). [24-“ClCholyltaurine was purchased from New England Nuclear (Boston, MA] and used without further purification. 24-‘C-Labeled CDCA, UDCA, deoxycholic acid (DCA), and nor-UC-tau were synthesized according to the method of Tserng et al. (27) and purified by thin-layer chromatography (TLC). Ileal perfusion. Fasting young adult male SpragueDawley rats (Charles River Laboratories, Wilmington, MA] with a weight range of 190-265 g were anesthetized with pentobarbital sodium (Nembutal, Abbott Laboratories, Chicago, IL), 70 mgikg, IP. The external jugular vein was cannulated using polyethylene tubing (PE 10; ID, 0.28 mm; OD, 0.61 mm; Clay Adams, Parsipanny, NJ), and anesthesia was maintained by a continuous IV infusion of a 2.5-mg/mL pentobarbital solution via an infusion pump at a rate of 0.03 mg/min. After laparotomy, the common bile duct was cannulated with polyethylene tubing (PE 10). A large-bore polyethylene tube (Pharmaseal, K50 extension tube, American Pharmaseal Co., Valencia, CA) was passed into the cecum through an incision in the proximal ascending colon. The tube was carefully pushed through the ileocecal valve so that the tip of the tube protruded no more than 5 mm into the distal terminal ileum. The entire small intestine was flushed clean using 60-80 mL of isotonic saline (at 37°C)

214

GASTROENTEROLOGY Vol. 100. No.

MARCUS ET AL

1

Table I. Chemical Structures of Conjugated Bile Acids

OOH

Nuclear substituents Preferred trivial name

Alternative trivial name

Natural taurine-conjugated bile acids Cholyltaurine Taurocholate Ursocholyltaurine Tauroursocholate TaurochenodeoxyChenodeoxycholyltaurine cholate UrsodeoxycholyltauTauroursodeoxyrine cholate Iso-ursodeoxycholylTauro-iso-ursodeoxytaurine cholate Deoxycholyltaurine Taurodeoxycholate LagodeoxycholyltauTaurolagodeoxyrine cholate Bile acids with modified side chain Nor-ursocholyltauTauro-nor-ursocholate rine Tauro-nor-ursodeoxyNor-ursodeoxycholyltaurine cholate Nor-ursodeoxyNor-tauro-nor-ursodecholyl-nor-taurine oxy-cholate

Abbreviation

R, 3-OH

Number of skeletal atoms Amino acid moiety

R, R, 7-OH l&OH

Bile acid side chain

(Y

cx

a

a

P

@z Taurine [tam

5 5

CDC-tau

Taurine [tau]

5

UDC-tau

Taurine [tau]

5

iso-UDC-tau DC-tau

Taurine [tau] Taurine [tau]

5 5

LDC-tau

Taurine [tau]

5

Taurine [tau]

4

Taurine [tau] Aminomethyl-sulfonic acid [ams]

4

C-tau UC-tau

nor-UC-tau

a

P

nor-UDC-tau

a

P

nor-UDC-ams

a

P

NOTE. A natural C,, bile acid is shown with its C, isopentanoic shown).

a

Taurine [tau]

side chain. The nor-bile

using a syringe. A small incision

was made 20 cm proximal to the ileocecal valve into the antimesenteric border, and a polyethylene tube (PE90; ID, 0.86 mm; OD, 1.27 mm; Clay Adams) was introduced into the lumen of the ileum and tied in place using a ligature that also prevented flow from the more proximal intestine from entering the perfused segment. A temperature probe was also inserted into the proximal ileal lumen. The abdominal wall was then closed carefully with surgical clips. The perfusate was pumped through a coil immersed in a thermostatted water bath so that it entered the animal with a temperature of 38 f 0.5”C as measured by the temperature probe. After the abdomen was closed, body temperature was maintained at 38 ? 0.5”C by means of a thermostatically controlled heating lamp connected to a rectal temperature probe. A piston-driven pump [Lab Pump Jr. Model RHSY, Fluid Metering Inc., Oyster Bay, NY) was used to perfuse the 20-cm test segment of ileum. The pulsatile flow rate was adjusted to 4 mL/min. Preliminary studies confirmed that at this flow rate absorption of the perfused bile salts was less

Amino acid moiety

4

acids have a C, isobutyric acid side chain (not

than 10% and that there was no apparent distention of the ileum. The absorption rate of cholyltaurine at low concentrations (1.0 mmol/L) or high concentrations (5.0 mmol/L) did not increase when the perfusion rate was increased to 10 mL/min, indicating that diffusion through the unstirred layer was not rate-limiting (cf. 28, 29). Each concentration of bile acid was perfused for 1 hour. Bile was collected at lo-minute intervals into preweighed tubes, and the volume of bile was determined gravimetritally. The last three collections of the hour were averaged because preliminary studies showed that a steady state was attained after 30 minutes. The perfusate consisted of an isotonic electrolyte-taurineconjugated bile acid solution. Each taurine-conjugated bile acid (0.25-16 mmol/L) was dissolved in an isotonic buffer of 130 mmol/L sodium chloride, 2 mmol/L calcium chloride dihydrate, and 25 mmol/L tris(hydroxymethy1) aminomethane (Trizma base, Sigma Chemical Co., St. Louis, MO), adjusted to pH 8 with 1 mol/L HCl. In experiments in which C-tau, DC-tau, CDC-tau, UDC-tau, and nor-UC-tau

January

AI3SORPTION OF CONJUGATED BILE ACIDS IN VIVO

1991

Table 2. Chromatographic

and Physicochemical

RRT by HPLC

Preferred trivial name Natural taurine-conjugated C-tau UC-tau CDC-tau UDC-tau Iso-UDC-tau DC-tau LDC-tau Bile acids with modified

Nor-UC-tau Nor-UDC-tau Nor-UDC-ams

Properties

&by TLCb

CMC (0.15 moVL Na’)

bile acids 0.46 0.23 0.70 0.33 0.22 0.82 0.22

0.23 0.28 0.39 0.41 0.42 0.37 0.43

10 40 1.4 4.3 8 1.5 1.9

side chain 0.07 0.25 0.24

0.24 0.40 0.44

80 12 20

“Relative retention time by HPLC to chenodeoxycholylglycine using a C,, octadecyl silane stationary phase and an isocratic elution with buffered methanol-water as described by Rossi et al. (20). *R, mobility relative to the front by adsorption chromatography on silica gel layers using an acidic system previously described for the separation of conjugated bile acids (25). “CMC, concentration within the narrow range of concentration in which bile acid anions progressively self-associate to form multimers. Values reported were previously obtained by Roda et al. using a maximum bubble pressure device (21) or obtained for this study by Jan Lillienau and K. J. Mysels using this same device (unpublished observations).

the ‘*C-labeled taurine-conjugated bile acid was added in tracer amounts to achieve a specific activity of IIOO-47,000 dpm/mmol. In the remaining experiments, no label was added. Each animal was perfused with only a single bile acid. In most experiments, three concentrations were used, in random order, each concentration being perfused for an hour. Perfusion of the ileum with CDC-tau or DC-tau for > 3 hours caused a progressive decrease in the bile acid transport rate; for these bile acids, only two doses per animal were given. In addition, as noted below, concentrations of DC-tau > 1.5 mmol/L and of CDC-tau > 3 mmol/L were toxic, resulting in death of the experimental animals. Therefore, the concentration range for CDC-tau and DC-tau was limited to 0.25-0.75 mmol/L. Each concentration of conjugated bile acid was tested three to five times. Histological study of the perfused ileum after 3 hours indicated no evidence of morphological damage except for the two toxic bile acids CDC-tau and DC-tau.

were perfused,

Analytical Methods

because these compounds were not available in radioactive form. For determining biliary bile acid radioactivity, 50 FL of with

10 mL of xylene-based

medium (Scintiverse E, Fisher Scientific, Tustin, CA) and counted using a liouid scintillation counter (Nuclear Chicago Mark I< E.D: Searle & Co., Des Plaines, IL] with automatic external standard for quench correction.

Data Analysis The length of intestine perfused exceeded the length of small intestine where most active transport takes place. Accordingly, the observed absorptive rate for the conjugated bile acids studied is believed to be only slightly less than that of the entire absorptive area of the small intestine. The absorption of conjugated bile acids from the large intestine the bile acid is also quite limited (12,14,15). Therefore, absorption rate was expressed as kmol/min ’kg body weight rather than per unit length of perfused intestine. The relationship between ileal bile acid concentration and recovery was assumed to fit the following relationship: Recovery = [[Bile Acid] T,,,)/(K, + [Bile Acid]). MichaelisMenten kinetics were used for convenience, but it is realized that two transport systems-one on the apical membrane and a second on the basolateral membrane-are involved in ileal bile acid transport (18). In addition, a small component of passive absorption could not be excluded (see Results). Micelle formation prevented measurement of a For other compounds, the true T,,, value for cholyltaurine. amount of chromatographically purified material available was insufficient to perfuse at concentrations several times the calculated value for K,, as would have been desirable (for example, a single perfusion with a 6-mmol/L solution used about 800 mg of material]. Best-fit coefficients of the recovery curve for each bile acid were obtained by pooling data from all animals studied. Analysis of covariance for iso-UDC-tau (the bile acid having the most animals with more data points than coefficients] showed no significant error reduction (P > 0.05) when the animals were considered separately.

Recovery curves for the different bile acids were compared using analysis of covariance. Absorption curves were analyzed using nonlinear regression; T,,, and Ic, values were calculated. The term T,,, has been used rather than V,,, because two transport systems are involved, and T,,, does not have mechanistic significance. For the two toxic dihydroxy bile acid conjugates, linear regression was used to calculate the relationship between absorption rate and concentration.

Results

The total biliary bile acid concentration in bile was measured using 8a-hydroxysteroid dehydrogenase (Sigma) as described by Turley and Dietschy (36). Standard curves were determined with the individual bile acid used, but the enzymatic reactivities of all of the da-hydroxy bile acids used in these studies were essentially identical to that of cholate. For iso-UDC-tau and LDC-tau HPLC (21) was used,

bile was mixed

215

scintillation

as micromoles per minuteThe T,,,, expressed kilogram of rat body weight, and K,,,, in millimoles per liter, are given for all bile acid conjugates in Table 3. Values for the quotient (T,,/K,,,) are summarized in Table 3; this quotient permits comparison of absorption rates in relation to concentration at concentrations well below the K,,,. Figures l-5 compare the transport rates of bile acids for groups of related conjugates. All bile acids were well transported at concentra-

216

MARCUS ET AL.

GASTROENTEROLOGY Vol. 100, No. 1

Table 3. Kinetic Parameters for Ileal Transport of Bile Acid Conjugates Range of concentration perfused (mmol/L)

Bile acid

Total no. of steady-state perfusions

Natural taurine-conjugated bile acids C-tau 0.25-10 UC-tau 0.25-12 CDC-tau 0.25-l 0.25-6 UDC-tau 0.25-l Iso-UDC-tau DC-tau 0.25-l LDC-tau 0.25-5 Bile acids with modified side chain Nor-UC-tau 1.0-10 Nor-UDC-tau 1.0-16 0.25-6 Nor-UDC-ams

CMC

T,,, (CL”)

K, (CL”1

T,,&,

(mmol/L)

(pmollkgmin)

(mmol/L)

(mLlkgmin)

26 25 13 23 14 10 14

10 40 1.4 4.3 8 1.5 1.9

12.9 (12.0-13.7) 9.6 (9.1-10.1)

6.7 (6.0-7.7) 6.5 (5.8-7.2) -

5.0 (4.2-5.7) 1.6 (1.1-2.0) -

3.1 (2.3-4.3) 0.7 (0.6-1.3) -

1.2 (1.0-1.4)

0.3 (0.2-0.5)

1.9 1.5 1.9* 1.6 2.3 1.6* 4.0

11 24 27

80 12 20

8.4 (8.0-8.9) 5.9 (5.3-6.6) 9.1 (8.5-9.6)

6.0 (5.4-6.7) 2.6 (1.7-3.7) 6.3 (5.8-7.0)

1.4 2.3 1.4

“95% confidence limits for coefficients. %alculated by linear regression. T_ value could not be obtained because of toxicity.

tions up to 0.5 mmol/L. Above this concentration, the transport of bile acids varied widely. The two lipophilic dihydroxy bile acids, CDC-tau and DC-tau, caused systemic toxicity and death when infused above concentrations of 3 and 1.5 mmol/L, respectively, which would correspond to an expected hepatic transport rate of 1.5-3 tJ,mol/min * kg. Because of this toxicity, accurate kinetic parameters for ileal transport of these bile acids could not be determined. However, the slope of the line relating absorption rate to concentration was 1.8 for DC-tau and 1.9 for CDC-tau (at concentrations I 0.75 mmol/L), suggesting that, in the absence of toxicity, ileal transport parameters of these bile acids would be similar to those of other bile acids (Figures 1 and 2).

7C 2

The remaining bile acids were nontoxic. Cholyltaurine, a naturally occurring bile acid in the rat, was transported to the greatest extent with a calculated T,,, of 12.7 kmol/min + kg (Figure 3). This value would correspond to an intraluminal concentration well above its CMC value of 10 mmol/L (21). Hence, the ileal transport of cholyltaurine may have been limited by micelle formation, because the monomer concentration of cholyltaurine increases only slightly at concentrations greater than its CMC (31). Ursocholyltaurine, which differs from C-tau in having a 7(3-hydroxy group, also showed similar saturation, but had a lower T,,, of 9.6 pmol/min * kg (P < 0.001; Figure 3); saturation of transport began at a concentration of lo-12 mmol/L, a concentration at which only

10 T

CDC-tau

0

0

l/DC-tau

0 LDC-tau C-tau

C-tau

al 1

G

OF 0

:

DC-tau

0

i

1

3

2 Bile

acid

4 cont..

5

6

mM

Figure 1. Relationship between intraluminal bile acid concentration in the perfused ileum and bile acid recovery in hepatic bile for CDC-tau and UDC-tau in the anesthetized rat with a biliary fistula. The values for C-tau are indicated by the d&redline.

‘1

co

0

1

2 Bile

3 acid

4 cont.,

5

6

mM

Figure 2. Relationship between intraluminal bile acid concentration in the perfused ileum and bile acid recovery in hepatic bile for DC-tau and LDC-tau in the anesthetized rat with a biliary fistula. The values for C-tau are indicated by a dashedline.

January 1991

ABSORPTION OF CONJUGATED BILE ACIDS IN VIVO

217

insufficient material was available to obtain an accurate estimate of T,,,; the estimated value was significantly lower than that of cholyltaurine (P = 0.02). Contribution of Passive Absorption

? ? C-tau

0 UC-tau 2

v nor-UC-tau

-tAY Oy.

G

0

2

4

6 Bile

6 acid

10 12 14 cone., mM

16

Figure 3. Relationship between intraluminal bile acid concentration in the perfused ileum and bile acid recovery in hepatic bile for C-tau, UC-tau, and nor-UC-tau in the anesthetized rat with a biliary fistula. The values for C-tau are indicated by a dashed line.

monomers would be expected to be present (Figure 1) because UC-tau’s CMC is 40 mmol/L. Figures 1 and 2 compare the transport of the naturally occurring toxic lipophilic dihydroxy bile acids with that of their corresponding hydrophilic p-hydroxy epimers. Ursodeoxycholyltaurine was moderately well transported with a T,,, of 5.0 pmol/min . kg. This transport rate was significantly slower than that of C-tau (P < 0.001). Lagodeoxycholyltaurine, the 12p epimer of DC-tau, had a T,,, of 1.2 tJ,mol/min * kg compared with C-tau (P < 0.001). Figure 4 compares the transport rates of three ursodeoxycholate (UDC) derivatives varying in the length of the side chain and indicates that side-chain length influences the ileal transport rate (P < 0.01). Ursodeoxycholyltaurine has 8 atoms (6 carbon plus 1 nitrogen plus 1 sulfur) in the side-chain skeleton; nor-UDC-tau has 7; and nor-UDC-ams has 6. All three homologues were well transported, but the homologue with the shortest side chain (nor-UDC-ams) was transported significantly more rapidly than UDC-tau (P < 0.001). Nor-ursocholyltaurine (C, side chain) was transported (T,,,, 8.4 pmol/min . kg) at a rate similar to that of UC-tau (C, side chain; T,,,, 9.6 Fmol/min * kg). At the concentration at which maximal bile acid transport was obtained, only monomers were present because the compound has a CMC of 80 mmol/L. Both ursocholyl conjugates were transported more slowly than C-tau (P < 0.001). Figure 5 compares the transport rate of UDC-tau with that of iso-UDC-tau, the taurine conjugate of its 36-hydroxy epimer. The unnatural hydrophilic bile acid (iso-UDC-tau) was transported slowly with an approximate T,,, of only 1.6 tJ,mol/min * kg. However,

Using the same technique, the absorption of dibromosulfophthalein, a dianionic organic anion with a molecular weight of 632, was examined to determine the extent of passive absorption; no absorption was detected. This compound is known to be efficiently eliminated in bile if administered IV (32). Additional evidence that little passive absorption (either transcellular or paracellular) occurred in these experiments was provided by the data for LDC-tau, which showed little absorption, even at a perfusate concentration of 5 mmol/L (Figure 2). A line connecting the origin and the recovery when the perfusate was at a concentration of 5 mmol/L indicates the limiting value for passive absorption in these experiments. Biotransformation

Of the nontoxic bile acids, C-tau, UC-tau, and nor-UC-tau were not biotransformed. Ursodeoxycholyl taurine, nor-UDC-tau, nor-UDC-ams, and UDCtau did not undergo substantial biotransformation, but small amounts of polar derivatives were formed, as is known to occur during hepatic transport. (Such changes are likely to be additional hydroxylation at the 6 or 7 positions as well as sulfation or ether glucuronidation of the 3hydroxyl group.) The only bile acid undergoing extensive biotransformation was

10

T 8

,

/-

t /

64

2t

’

/ ’

;/ c T 4

Bile

6

C-tau

3

? ? UDC-tau

0 nor-UDC-tau

J

2

/--

/’

T

v nor-UDC-ams

01 0

,

i3

Acid

10 cont.,

12

14

16

mM

Figure 4. Relationship between intraluminal bile acid concentration in the perfused ileum and bile acid recovery in hepatic bile for UDC-tau, nor-UDC-tau, and nor-UDC-ams in the anesthetized rat with a biliary fistula. The values for C-tau are indicated by a dashed line.

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ABSORPTION

1991

rodent ileum (15,41). Whether lengthening the side chain influences ileal transport is unknown, but a tripeptide conjugate of cholic acid was reported to be well transported by the rat ileum (42). Whether these structure-transport-toxicity relationships apply to species other than the rat is unknown. In humans, based on intestinal perfusion studies of ileal bile acid absorption, trihydroxy bile acids have a greater absorption rate for a given intraluminal concentration than dihydroxy conjugated bile acids (141, as was observed in these studies. Rates of ileal transport are much lower in humans than in rats. In healthy subjects, the ileal absorption rate during digestion averages 0.3-1.0 kmol/min * kg based on hepatic secretion rate (43-45), a value about one fifth that reported for the rat (l-5). The T,,, value for bile acid transport by the total ileum is unknown in humans. For hepatic transport of bile acids, structure-transport relationships have been shown to vary among rodents, with the highest values for T,,, being observed for those bile acids that are endogenous to a given species (46).

OF CONJUGATED

BILE ACIDS IN VIVO

219

rates can be found to vary from one twelfth to greater than that of hepatic T,,, for the bile acids. One bile acid that had a strikingly different T,,, for transport between liver and ileum was LDC-tau: the T,,, for hepatic transport was at least 12 times that of ileal transport. Presumably, the T,,, values for ileal transport of CDC-tau and DC-tau are greater than the respective T,,, values for hepatic transport. Such a comparison of hepatic and ileal transport rates cannot be made in humans because the hepatic T,,, for conjugated bile acids has never been determined, presumably because of concern of the safety of such an experiment. In conclusion, these studies define the maximal transport capacity of the rat ileum for taurineconjugated bile acids; they indicate that the ileal transport system in the rat is of low affinity and high capacity for taurine conjugates of hydrophilic bile acids, and they show that both nuclear substituents and side-chain length influence the transport rate of taurine-conjugated bile acids.

Appendix Physiological Implications

The intraluminal concentration of bile acids in the distal ileum of the rat is about l-3 mmol/L (47), indicating that absorption should be occurring at concentrations below the k, at least for C-tau. Simultaneous water and electrolyte absorption by the terminal ileum should increase bile acid concentration and hence the total amount of bile acids absorbed. Comparison

of Ileal With Hepa tic Transport

Table 4 compares the values obtained in this study for ileal T,,, with published values (22,48-50) for hepatic T,,, in the rat. Maximal ileal transport

Table 4. Comparison of Maximal Rates of Ileal and Hepatic Transport Rates of Some Taurine-Conjugated Bile Acids in the Rat T mai Bile acid

Ileal

Natural taurine-conjugated 13 C-tau 10 UC-tau 5 UDC-tau 2 LDC-tau Bile acids with modified 8 Nor-UC-tau 12 Nor-UDC-tau

Hepatic bile acids 15 8 32 >25 side chain 32 18

Ratio: ileal/hepatic

Reference

0.86 1.25 0.16 0.08

49 50 51 23

0.25 0.66

0 h

“Unpublished observations (J.L.) performed for this paper. Hepatic T,,, was measured essentially as described by Kitani et al. (50). %npublished observations of Y. B. Yoon in this laboratory using methodology previously reported (49).

Cholyltaurine, 2-[[3a,7a,l2ol-trihydroxy-24-oxo-5(3cholan-24-yllamino] ethanesulfonic acid 2-[[3o,7l3,12ol-trihydroxy-24-oxoUrsocholyltaurine, 5(3-cholan-24-yl]amino]ethanesulfonic acid Chenodeoxycholyltaurine, 2[[3a,7a-dihydroxy-240x0-5P-cholan-24yl]amino]ethanesulfonic acid Deoxycholyltaurine, 2-[[3c~,12o-dihydroxy-24oxo-5(3cholan-24-yl]amino]ethanesulfonic acid Ursodeoxycholyltaurine, 2-[[3a,7(3-dihydroxy-24oxo5P-cholan-24-yl]amino]ethanesulfonic acid Nor-ursodeoxycholyltaurine, 2-[[3a,7(3-dihydroxy-24nor-23-oxo-5(.$cholan-23yllaminolethanesulfonic acid Iso-ursodeoxycholyltaurine, 2-[[3(3,7(3-dihydroxy-24oxo-5(3-cholan-24-yl]amino]ethanesulfonic acid 2[[3a,l2(9dihydroxy-24Lagodeoxycholyltaurine, oxo-5(3-cholan-24yl]amino]ethanesulfonic acid Nor-ursodeoxycholyl-nor-taurine, l-[[3a,7(+dihydroxy-24-nor-23-oxo-5P-cholan-23-yl]amino]methanesulfonic acid 2-[[3a,7($12a-trihydroxy-24Nor-ursocholyltaurine, nor-23-oxo-5(3-cholan-23-yl]amino]ethanesulfonic acid

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43. van Berge Henegouwen GP, Hofmann AF. Nocturnal gallbladder storage and emptying in gallstone patients and healthy subjects. Gastroenterology 1978;75:879-885. 44. Leiss 0, von Bergmann K. Comparison of biliary lipid secretion in non-obese cholesterol gallstone patients with normal, young, male volunteers. Klin Wschr 1985;63:1163-1169. 45. Everson GT, Lawson MJ, McKinlay C, Showalter R, Kern F Jr. Gallbladder and small intestinal regulation of biliary lipid secretion during intraduodenal infusion of standard stimuli. J Clin Invest 1983;71:596-603. 46. Aldini R, Roda A, Morselli Labate AM, Grigolo B, Simoni P, Roda E, Barbara L. Species differences of the hepatic uptake of bile acids. Ital J Gastroenterol 1986;19:1-4. 47. Dietschy JM. Mechanism for the intestinal absorption of bile acids. J Lipid Res 1968;9:297-309. 48. O’Maille ERL, Hofmann AF. Relatively high biliary secretory maximum for non-micelle-forming bile acid: possible significance for mechanism of secretion. Q J Exptl Physiol 1986;71: 475-482. 49. Yoon YB, Hagey LR, Hofmann AF, Gurantz D, Michelotti EL, Steinbach JH. Effect of side-chain shortening on the physiological properties of bile acids: hepatic transport and effect on

Received December 27, 1989. Accepted July 13, 1990. Address requests for reprints to: Alan F. Hofmann, M.D., Ph.D., Division of Gastroenterology, 0813, Department of Medicine, University of California, San Diego, La Jolla, California 92093. Supported in part by National Institutes of Health grants DK 21506 and DK 32130 as well as grants-in-aid from the Diamalt Company, Raubling, Germany, the Falk Foundation, Freiburg, Germany, and Medstone International, Inc., Irvine, California. This work appeared previously in abstract form (Gastroenterology 1989;96:A230). Dr. Marcus was a Medical Research Council (United Kingdom) Travelling Fellow. His present address is: Department of Medicine, Palo Alto VA Hospital, Palo Alto, California. Y. Xia is presently a graduate student in the Department of Physiology, Ohio State University, Columbus, Ohio. J. Lillienau is a visiting fellow supported by the Swedish Medical Council. M. A. Angellotti is presently a member of the Institute of Scientific Chemistry, Faculty of Pharmacy, University of Bologna, Bologna, Italy. Dr. Schmassmann was a fellow of the Swiss Research Council. His present address is: Medizinische Klinik, Kantonsspital Aarau, 5999 Aarau, Switzerland.