Environmental Health PerspectivesVol. 33, pp. 79-90, 1979

Properties and Biological Significance ofthe leal Bile Salt Transport Systemby Leon Lack*

The properties of a specific transport system for bile salts, which is located in the ileum of the smallintestine are described. The system operates by a sodium ion cotransport mechanism, and it functions inmaintaining a normal enterohepatic circulation of bile salts. Analysis of structure-activity data allows us todepict our hypothesis for the interaction of the bile salt and Na with the membranal recognition site of thistransport system. The sequellae ofmetabolic disorders which can arise following disease or surgical ablationof the ileal region of the intestine which result in an interrupted bile salt enterohepatic circulation aredescribed. We suggest that these findings hold interest to toxicologists, since it is not beyond reason thattoxic agents might exist which impair the function ofthis transport system specifically or which could poisonthe ileal mucosal cell. Such agents might be detected by the presence of some of the described metabolicdisorders. Finally, we discuss the ileal transport ofthe sulfated esters of bile salts and the possibility that thismight relate to that aspect of detoxification pertaining to their enhanced excretion.

Bile salts are detergentlike steroid derivativeswhich are synthesized in the liver from cholesterol.Their role in the digestive process has long beenrecognized. Bile salts are utilized within the intesti-nal lumen to emulsify lipid in order to facilitate itsdigestion. They function additionally by dispersingthe products of lipid digestion, i.e., long-chain fattyacids and 2-monoglycerides, into micelles, thus al-lowing for their more efficient absorption. In theabsence of bile salts the absorption of dietary lipidmay be impaired by as much as 50%. The absorptionof cholesterol and fat-soluble vitamins (A, E, and K)does not take place in the absence of bile salts. Bilesalts are also required for the optimal function ofpancreatic lipase. In addition, they have a criticalrole in maintaining cholesterol in a micellar stateduring the elaboration and storage of bile. It is be-lieved that cholesterol gall stones can occur when aphysiological imbalance between bile cholesteroland bile salts arises.These various functions apparently require large

quantities of bile salts. Hepatic secretion in man inthe order of 30 g/day is about six times the total poolsize and many times the rate of normal biosynthesis.

*Department of Pharmacology, Box 3185, Duke UniversityMedical Center, Durham, North Carolina 27710.

Enterohepatic circulation of the bile salts may beregarded as a mechanism for the conservation ofthese physiologically important substances. Figure 1depicts this process schematically for man.The pool size can be approximated as 3-5 g. In the

normal preprandial state, the bulk of this materialwould be found in the gall bladder. Following thedischarge of material from the gall bladder, the bilesalts enter the intestine to become intimately as-sociated with the foodstuffs in order for them tofunction in the manner already alluded to. Sub-sequent to the absorption of lipids from the proximalregion of the small intestine, the bile salts are ab-sorbed predominantly but not exclusively from theileum. They then return to the liver by the portalroute to be resecreted into the bile in order to con-tinue the digestive absorptive process. Thus, duringthe digestion and absorption ofa single meal this poolmay circulate two or three times. The small amountof material escaping enterohepatic circulation (ap-proximately 0.3 g/day) is restored by an equal con-version of cholesterol to bile salts by the liver. Iwould like to interject that this conversion in the liverrepresents a very real homeostatic process. Thus, iffor some reason the enterohepatic process is inter-rupted for example, by a bile duct fistula or bile saltmalabsorption, the liver increases the rate ofconver-sion ofcholesterol to bile salt by as much as sixfold in

December 1979 79

of the intestine. It can be seen that only those sacsprepared from the distal region can generate uphillmovement from the mucosal to the serosal side of thegut wall, as evidenced by the final serosal to mucosalratios greater than 1. This process is inhibited bysodium azide, dinitrophenol, and anoxia. Similar re-sults were obtained when this type of experimentwas performed with intestine sacs made from guineapigs, hamsters, mice, spider monkeys, pigeons, andchickens (6).Much of our in vitro work describing the nature of

this process has been done with everted gut sacsprepared from guinea pigs. However, whenever pos-sible, the specific in vitro observation in questionwas checked or documented with in vivo guinea pigmodels. The in vivo model involved perfusion of ilealorjejunal intestinal segments in sito in animals bear-ing common bile duct fistulae. It was possible toquantitate transmucosal movement of the test bilesalts or derivatives by measuring the rate of theirappearance in the secreted bile (7) which was col-lected via the cannulla in the bile duct fistula (seeFig. 3).Employing these models we have collected con-

siderable data concerning structure-activity re-

FIGuRE 1. Schematic representation of the enterohepatic circula-tion of bile salts. The broken line represents bile salts whichhave been modified by bacterial action and reabsorbed fromthe colon by passive diffusion (28).

an attempt to maintain a normal bile salt pool. Theseaspects of bile salt physiology have been thoroughlyreviewed (1-3).As far as the intraintestinal events of lipid diges-

tion are concerned, I would like to point out that thisanatomical arrangement, where lipids are absorbedfrom the proximal region ofthe small bowel while thebile salts leave the intestine from the more distalregion, could represent an adaptation that wouldallow optimal concentration of the detergent bilesalts in the proximal region of the gut.Some time ago we demonstrated that an active

transport system for bile salt exists in the intestineand that this system exists only in the ileal region (4).These early in vitro observations were made with theeverted gut sac preparation of Wilson and Wiseman(5). Some of these findings are shown in Figure 2.The everted sacs were prepared from segments of

the rat's small bowel. Each segment was one quarter

"eno0

0

c t

0

C0

0-C

0

S.-c0

I._

-J0

I

C.)

4

SegmentsProximal -a 1Distal

(20)8 _

6

4

2

(5) (5) L

Ncli0

C.)

IC)Nz

U')0

Px2

IcqI

z4

(8)' (8)

3iwi--IHsu-I(8)

C L _ _- i E,, ,,,.K S |1 2 3 4 --Segment 4 only-'-

FIGuRE 2. Transport of taurocholate by everted sacs of rat intes-tine. Each segment was one quarter the length of the smallintestine. The numbers in parenthesis represent the number ofanimals in each group. The bars give ± S.E. DNP = 2,4-dinitrophenol (4).

Environmental Health Perspectives80

FIGuRE 3. Illustration of the apparatus used for perfusing guineapig ileum in vivo (4).

lationships and electrolyte requirements. Some ofthese findings will be reviewed in this communica-tion. In addition, we will describe our hypotheticalmodel for (bile salt) substrate- carrier interactionsand then relate these findings and proposals to adiscussion of those relevant aspects that might havepotential interest to the toxicologist.

22

128v 23 26

24

H-HO 5

4 6CHOLESTEROL

C-O-HHO' H 'OH ° HO

CHENODEOXYCHOLIC ACID

OH

t LC-NHCH2COO-HO-'OH O

GLYCOCHOLATE

OH

><C-OH

CHOLIC ACID

C-OH

HS ^ CHOLANIC ACI D

OH

IN C-NHCH2CH2SO2O-HO- H '-OH 0

TAUROCHOLATE

5 CHOLANIC-AOH

5 a CHOLANIC ACID

From the chemical structures of two importantunconjugated bile acids - cholic acid andchenodeoxycholic acid - the chemical derivationfrom cholesterol is apparent. The biochemical path-ways are rather complex and have been reviewed(1-3). Prior to being secreted into the bile these sub-stances are conjugated with either taurine or glycineor a mixture of both. In man, the ratio of glycine totaurine conjugated bile salts is, under normal cir-cumstances, about 3:1.

In our early structure activity studies we ascer-tained that no particular hydroxyl group on the bilesalt was essential for its active transport (Fig. 4). Thethree derivatives of taurocholanic acid - 3,12-di-hydroxy-, 3,7-dihydroxy-, and 7,12-dihydroxy -can all be transported by everted gut sacs made fromthe ileum. In essence, we have taken taurocholatethe 3, 7, 12-trihydroxy compound whose transportwas already demonstrated (4) - and selectively re-moved a specific hydroxyl group from each position

_ 8 7JALC-NCH2CH2SO20H8

6

co 4- C\Cr _2O

SEG. I SEG. 2 SEG. 3 SEG. 4ProximaI Distal

FIGURE 4. Transport ofvarious bile salts by everted sacs ofguineapig small intestine. Figure is a composite of previous data (8).

and still maintained transport activity. The triketocompound is not a natural substance. Here one canenvision taurocholate with its three hydroxyl groupsoxidized to three keto groups. This compound com-pletely devoid of hydroxyl substituents still retainssome residual capacity to act as a substrate for thistransport system. It should be noted that the forma-tion of the triketo compound results in the stereo-chemical distortion of the normal shape of thecholanic acid steroid.When we studied the mutual inhibition between

the di- and trihydroxy compounds we ascertained onthe basis of in vivo and in vitro studies that (1) di-hydroxy bile salts are better inhibitors than thetrihydroxylated compounds; (2) trihydroxylatedcompounds are more readily inhibited than the di-hydroxylated substances; (3) the triketo compounds(which I have mentioned has a distorted steroidstructure) are the poorest inhibitors and the com-pound most readily inhibited (7, 8).Thus, while the transport system does not abso-

lutely require a specific hydroxyl group for inter-action, the hydroxyl groups do influence transportactivity, in that there appears to be an inverse re-lationship between the number of hydroxyl groupson the steroid nucleus and the apparent affinity be-tween substrate and transport system as indicated bythe mutual inhibition studies. This seeming anomalyremains to be explained and would have to be con-sidered in any hypothesis concerning substrate-car-rier interaction.At physiological pH the natural bile salts all pos-

sess a single negative charge on the side chain. Ourstructure activity studies would appear to indicate

December 1979 81

that this structural element - a single negativecharge on the side chain - was essential for optimaltransport. Figure 5A compares the in vitro transportof taurocholate with two dibasic or dianionic deriva-tives. It can be seen that the dibasic substances werepoorly transported when compared with theirnatural analog. Figure 5B compares the effects ofaltering the pH of the incubation media on this pro-cess. Glycocholate is the natural analog, and thecarboxymethyl derivative cholylaspartate is the testsubstance. At lower pH the transport ofglycocholatedecreases, while that of cholylaspartate increases inabsolute terms and relative to that of glycocholate.Since one would expect that more of the singlycharged species of cholyaspartate would exist atlower pH, its enhanced uphill movement with lowerpH would agree with the concept that a single nega-tive charge on the side chain is a critical structuralfactor. These findings were confirmed with similarstudies in in vivo preparations (10).

It may be of interest to consider reasons why asubstrate bearing two negative charges in this regionof the molecule should not be transported. A possi-ble explanation would be that during normal trans-port the bile salt interacts with an active site bearinga positive charge. If a negative charge existed in theregion of this active site, sufficient repulsion mightexist between the transport system and the secondnegative charge on the unnatural substrate to pre-vent transport. One may speculate further concern-ing the nature of this proposed negative charge in theregion of the active site. If under normal conditions itreacted with sodium, it might be functional in thetransport process, since sodium ions are necessaryfor the transport of bile salts (11, 12).To accommodate the above speculation we pro-

pose that the initial interaction of the bile salt sub-strate and sodium with the membrane recognitionsite or carrier occurs in a manner depicted in Fig-ure 6.Three components of interaction are involved: (a)

an interaction of the steroid part of the bile salt andthe carrier; (b) a coulombic interaction between thenegatively charged side chain and a positively

Table 1. Metabolic disorders resulting from ileal disease.

Steatorrhea (>5.5 g/24 hr with intake of 70-90 g LCT/24 hr)Watery diarrhea (>200 g/24 hr, Na > 7 meq/24 hr, K > 20 meq/24

hr)Hypercholesterolemia or lower range of normalRelative deficiency of taurine (glycine: taurine of conjugated bile

salts considerably > 3:1)Bile supersaturated with cholesterol (gallstones)Oxaluria (nephrolithiasis)B12 deficiency (abnormal 2nd stage Schilling test)

-amii

c1

2u

co

.S-

IC0

0CC)

SU.

pH 7.62 pH 6.04

Substrate Final Serosal/Mucosal Ratio*

o 0R - C - N-CH2CH2-S -0 15.3

0

Taurocholate.

o 0R - C - NH CH2CH2-P<0 (-) 1.8

0

R - C - NH CH2-COOIC(COCH2-C00

1.2

FIGURE 5. Effect ofpH on transport of glycocholate and cholylas-partate (9) and comparison of taurocholate transport with di-basic analogs.

charged element in the membrane or carrier (desig-nated cationic site), and (c) a closely positionedfunction of negative charge on the carrier which caninteract with the sodium ions. Furthermore, we pro-pose that these three components of interactionfunction cooperatively, and that this structure orrecognition site resides within a hydrophobic creviceor pouch in the membrane. This complex of specula-tions could explain the results of our mutual inhibi-tion studies. Thus, even though no specific hydroxylgroup is required for a critical interaction betweencarier and substrate, those compounds with fewerhydroxyl groups would have better accessibility tothe hydrophobic space; in mutual inhibition studies,for example, between di- and trihydroxylated sub-strates, more of the dihydroxy compound would

Environmental Health Perspectives82

CHOLICACID

- . ,3

it

-10 a

UI- N

* 3 sites interact cooperatively.* All this takes place in a hydrophobic

space or cleft.FIGURE 6. Hypothetical scheme for the interaction of a bile salt

with its recognition site in the membrane. Also shown are thevarious ways of depicting the cholic acid moiety. The rep-resentational structures of cholic acid shown on left weretaken from the literature (1).

partition into this region and perform as a betterinhibitor. However, the oil/water partition sol-ubilities of the triketo compounds lie between thosefor their trihydroxy and dihydroxy analogs. Yet theyare the weakest inhibitors and the compounds mostreadily inhibited. We have attributed this to the factthat the already stated coplanarity requirements ofthe three keto groups have distorted the regularcholestane configuration common to the natural bilesalts, and one of the components of interactionnamely that between the membrane carrier and thesteroid moiety is not optimal.Such a complex hypothesis would dictate predic-

tions which are testable. For example, these specu-lates would predict that the Na requirements fortransport of the triketo bile salt substrate be greaterthan that for its natural analog. That this is so isshown in Figure 7. These are in vitro transportstudies. The incubations involved the usual Krebs-Ringer buffers with varying amounts of NaCl re-placed by isoosmotic equivalents of mannitol. As wedecrease the concentration of Na in the incubationsinvolving taurocholate, there is a significant but nottoo dramatic decrease in transport. Note that at 30

,,uu 100%

E- 90%E -0

S 160 -5: \~~~~~~~75%

o 120

0 100%80

40

140 90 60 30Na (mM)

FIGuRE 7. Comparison of the in vitro transport of taurocholatewith taurodehydrocholate (12) in media of different Na+ con-centrations. (-) transport of taurocholate (each point is themean + SEM of eight gut sacs); (0) transport of taurodehy-drocholate (each point is the mean + SEM of 16 gut sacs.Solutions were Krebs-Ringer bicarbonate with differentamounts of Na+. Mannitol was used as the osmotic replace-ment for NaCl. Concentration of substrate 28 nmole/ml.

mM Na+ transport is still 75% of control values.However at comparable Na levels, transport of thedistroted triketo analog of taurocholate is inhibitedby 75%. Also shown are the double reciprocal plotsof this data. Reference to the intercepts of theabscissa would show that the apparent affinity for Naby the transport system is much greater in the pres-ence of taurocholate than in the presence of thetriketo analog.The proposition of cooperativity would require

that mutual inhibition studies between taurodehy-drocholate and taurocholate demonstrate that tauro-dehydrocholate would function as a better inhibitorat higher Na ion concentration than at lower Nalevels. As one lowers the Na concentration, the in-teraction of the distorted triketo compound with thetransport system would decrease more than that ofthe natural compound and therefore act as a lesscapable inhibitor. This was found to be the case (12).

December 1979 83

The hypothetical scheme would predict that bilesalts modified in a manner such that there be nocharge on the side chain would still be capable ofinteracting with the transport site by virtue of thesteroid recognition component but that uphill trans-port should be depressed dramatically because thecoulombic interaction and the cationic membranesite could not take place. When such compoundswere synthesized and tested (13), we were able todemonstrate interaction as evidenced by the fact thatin vivo studies demonstrated preferential ileal ab-sorption. In addition, these compounds could inhibitin vitro bile salt transport in a manner that would beexpected from our earlier mutual inhibition studieswith the natural bile salts; i.e., the fewer the hy-droxyl groups, the better these compounds functionas inhibitors. In addition, the triketo analog iswithout effect. Uphill transport (against a concen-tration gradient) by everted gut sacs is either minimalor not observed, depending on the analog tested.Figure 8 demonstrates this transport with our mostactive analog, cholyl NPG. It is apparent that thisobserved transport is much less than that shown bythe natural congener, taurocholate. The proposal forcooperativity between the various sites would pre-dict that the transport of cholyl NPG would be moresensitive to Na ion depletion than its anionic analog.This too, has been demonstrated.

Cholyl NPG Touro -CH3 0 OH H cholote

QH CH-CHC2C-NHCHCH02

HO' -OH-A Mucosol 15 43b 630 230Compartment 2 X3 -. *n moles 2 2 ± 1 1

10E

400()f3F_ __

2j

1 2 3 4 Seg4Proximal - Distll

FIGURE 8. Transport of an uncharged bile salt derivative byeverted gut sacs prepared from different regions of the guineapig small bowel. Also shown is the transport oftaurocholate inparallel incubation of gut sacs from distal small bowel. (13).

If the proposed scheme for interaction were cor-rect, one would expect that positively charged orcationic analogs might not be transported at all. If thesteroid moiety of the derivative could still interact atthe steroid recognition site, then such compoundswould act as refractory substrates, and inhibition ofthe transport of natural bile salts could take place.Furthermore, the proposal would insist that theorder of inhibition of bile salt transport by the posi-tively charged derivatives follow the same order ob-served in the mutual inhibition studies, i.e., thecationic derivative with one hydroxyl group wouldbe a better inhibitor than those derivatives with twohydroxyl groups. The trihydroxy derivative wouldbe even less effective as an inhibitor and, of course,the triketo compounds even less potent. This orderof inhibition was observed in in vitro and in vivostudies (14).The proposal would suggest that the cotransport of

the Na cation and the bile salt anion from the lumenof the intestine across the ileal brush border mem-brane into the mucosal cell might be an electroneu-tral process. In other words, since the loaded carrieris depicted as neutral, transmembrane movement ofthe bile salt could be to a great extent, if not com-pletely independent of the nature of the anion in theincubation media. This would be in contrast to theknown transport processes involving glucose (15).Vesicles were prepared from intestinal brush bor-

der membranes obtained from guinea pig ileums andjejunum. It was possible to demonstrate enhancedtaurocholate uptake by vesicles made from ileal tis-sue which was dependent on the presence of a gra-dient of Na ions. Vesicles made from jejunal tissuedid not demonstrate such activity. These preliminarydata are demonstrated in Figure 9. Proximal or distalvesicles were incubated with mannitol and 14C-taurocholate. At the point indicated by the arrow,solutions of NaCI (Fig. 9A) were added containingthe "4C-taurocholate at the same concentration asthat in the incubation media. Note that there is anovershoot ofNa taurocholate uptake with ileal vesi-cles which presumably can be maintained until theNa activity inside the vesicles equals that on theoutside. With proximal vesicles the increase in up-take is modest, presumably reflecting the swelling ofvesicles following the diffusion of electrolyte. Thedata in Figures 9E and 9F demonstrate that neitherKCI or LiCI can replace NaCI. However, NaCNS,Na isethionate, or Na2SO4 can effectively replaceNaCI. Thus, the magnitude of the overshootphenomenon is not altered when the chloride ion isreplaced by a more permeant anion (Fig. 9B) or lesspermeant anions (Figs. 9C, 9D).

Finally, this model might have something to say

Environmental Health Perspectives84

C=

a) -

-~ 0

0-

) E

C, Woa

c() a)0)-a

C3C)

2 2.5 3.5 6 1 22.5 3.5 6Minutes

FIGURE 9. Uptake of 24-[U4C]-taurocholate by brush border membrane vesicles in the presence of experimental electrolytes. Bufferedelectrolyte solutions were added at the times indicated. Each value represents the coverage offour to eight experiments. Also shownare the standard errors of the means (16).

concerning the position of the second negativecharge which, as we mentioned, appears to aborttransport when introduced on the side chain. Thus,as one moves the second potential negative groupaway from the side chain area where the coulombicinteractions are presumed to occur, the questionarises whether substrate-carrier interaction couldmore likely occur. We will have something to sayabout this when we discuss the effects of bile saltsulfation and its relevance to toxicity. Let us proceedto discuss the physiological implications of thistransport system which can move bile salts out of thelumen into the portal circulation and which is presentonly in the ileum. There is no doubt that absorptionof bile salts can, to some degree, take place by pas-sive fluxes along the entire length of the small andlarge intestine. We will not discuss the discussionsthat are current concerning the quantitative role that

December 1979

these processes contribute to the overall entero-hepatic circulation. Certainly these (passive) pro-cesses are real, and furthermore, the tendency forsuch diffusion increases as the number of hydroxylgroups decreases. We will see that this becomes avery real problem when we discuss the enterohepaticcirculation of the monohydroxy bile salts oflithocholic acid, a secondary bile salt with tox-icological implications. However, as far as the ilealtransport is concerned, it is generally accepted thatthe removal of the ileum effectively interrupts theenterohepatic circulation of bile salts. This is in con-trast to the removal of proximal regions of the intes-tine, where it can be demonstrated that the biologicalhalf-life of the bile salt pool is minimally affected.With the loss of bile salt enterohepatic circulation

following ileal resection, the hepatic feedback mech-anism makes an attempt at compensation by en-

85

hancing the daily conversion of cholesterol to bilesalts by several fold. In this manner, enhancedamounts of bile salts now enter the colon. This wasfirst demonstrated by Weiner, Playoust, and Lackwith surgically prepared dogs at Johns Hopkins (17).Following my arrival at Duke, I had the opportunityto collaborate with Dr. Tyor in the G.I. Division ofthe Department of Medicine and we found that thesame pertained to patients with ileal disease or withpatients with ileal resection performed as conse-quence to ileal disease (18, 19).Surgical removal of the ileum for certain disease

states is widely practiced. Certain sequellae havebeen recognized to occur as a consequence. Somemetabolic disorders resulting from lack of ileumfunction are steatorrhea (> 5.5 g/24 hr with intake of70-90 g LCT/24 hr); watery diarrhea (> 200 g/24 hr,Na > 7 meg/24 hr, K > 20 meg/24 hr); hypocholes-terolemia or lower range of normal; relative defi-ciency of taurine (glycine: taurine of conjugated bilesalts considerably less than 3:1); bile supersaturatedwith cholesterol (gallstones); Oxaluria (nephro-lithiasis); and B12 deficiency (abnormal 2nd stageSchilling test).

Steatorrhea or impaired lipid absorption stemsfrom the fact that in the absence of ileal functionrecirculation ofbile salts during the course ofa day isinterrupted. In spite ofenhanced biosynthesis of bilesalts, after the first meal following the overnight fast,adequate hepatic bile salt secretion cannot be main-tained.Without a functioning ileal bile salt transport sys-

tem the amount of bile salts daily entering the colonis increased. Enteric bacteria have the property ofdeconjugating the bile salts and modifying the steroidstructure forming secondary bile salts. An importantone is deoxycholic acid derived from the 7-dehy-droxylation of cholic acid. The increased levels ofunconjugated dihydroxy bile acids have the propertyof inhibiting the Na K ATPase of the colon. With theconsequent decreased levels of electrolytes andwater absorption one very frequently sees a waterydiarrhea (20, 21).Hypocholesterolemia would appear to be due to

the increased drainage on the cholesterol pool fol-lowing the enhanced conversion of cholesterol tobile salts.Man is a species whose bile salt pool consists of

glycine and taurine conjugates in an approximateratio of 3: 1. Taurine is biosynthesized by the liverfrom cysteine. With enhanced biosynthesis follow-ing the loss of ileal function greater proportions ofbile salts are elaborated as glycine conjugates. Thisenhanced glycine to taurine ratio is primarily ofhepatic origin, probably reflecting inadequate avail-

ability of hepatic taurine from cysteine (22, 23).In the absence of adequate amounts of bile salts in

the elaborated bile one obtains a biological imbal-ance with a tendency for the cholesterol to precipi-tate out. Following his two-year visit to our labora-tory, Dr. Ken Heaton returned to England and did aretrospective study and ascertained that patientswho had their ileums removed had a higher thannormal incidence of gallstones (24). These enhancedstatistics became more apparent with greater timelapse; following resection the incidence ofgallstonesincreased.With ileal disease an increase of renal stones oc-

curs. These stones more often than not are of thecalcium oxalate type. Upon removal ofthe ileum onecan obtain hyperoxaluria (25).Two factors are probably involved here: with the

steatorrhea following loss of ileal function, increasedamounts offatty acids remain in the intestine and areexcreted in the feces. Fatty acids have a tendency toreact with Ca2+ in the intestinal contents to forminsoluble Ca soaps. It would appear that Ca2+ nor-mally reacts with dietary oxalate as a means ofinhibiting oxalate absorption. The decreased avail-ability of free Ca2+ in the intestine following theformation of Ca soaps could result in enhanced oxa-late absorption. In addition, it has been shown thatthe excess secondary bile salts affect the permeabil-ity of the large bowel to oxalate ions, and this toocould be a contributing factor (26).The B12 deficiency is not related to the loss of the

bile salt transport system but to the fact that thespecialized system for absorbing the B12 intrinsicfactor complex also exists exclusively in the ileum.Recently two cases were reported by Heubi et al.

(27) of children showing some of these disordersand this led the investigators to suspect that thesechildren had a genetic deficiency of the ileal bile salttransport system. Their uptake studies with biopsytissue would appear to indicate that this deficiencydoes indeed exist. Interestingly enough, they reportthat B12 absorption was normal.Metabolic disorders listed above should be of po-

tential interest to the toxicologist. We suggest thatshould an incidence of intoxication include any ofthese disorders one ought to think in terms of ilealfunction.

I have already alluded to the phenomonon that bileacids can be modified by intestinal bacteria to form agroup of substances referred to as secondary bilesalts. Lithocholic acid is a monohydroxylated bileacid that is formed in the intestine from conjugatedchenodeoxycholate compounds as a consequence ofbacterial removal of the glycine or taurine moietyand the OH group in the 7 -a position (Fig. 10). It can

Environmental Health Perspectives86

lItestine

Lithocholote

Gcoihcol HC-N-CH2-COO.l

Glycolithocholole

H0XSH

Glycolithodholote-3-e-Sulfole

I CHCo-N~~~C-N-H2-CH2-S03HAll OH grOups c wefiguretiw HO"3SOK.All ON roups a ofllurtelos DOTewrlhocholote-3-e-SlifOte

FIGuRE 10. Metabolic transformation of glycocholic acid during enterohepatic circulation. A similarsequence is operative for taurocholic acid. Glycine and taurine conjugates ofchenodeoxycholic acid,which have OH groups at the 3-a and 7-a-positions also undergo deconjugation and 7-a-dehydroxylation (28).

then return to the liver for further processing.Lithocholate acid and its taurine and glycine conju-gates have been implicated in a variety of tox-icological processes which were observed by severalinvestigators in a number of experimental animalmodels. These include cirrhosis of the liver, bile ducthyperplasia, and gallstone formation. Following in-tramuscular injection in man, such lithocholateshave been reported to cause local inflammation,malaise, and fever. This subject has been reviewedby Palmer (29).

In 1967 Palmer reported that the liver was capableof sulfating lithocholate bile salts (30, 31). This was avery important observation, since it represented thedescription of a new metabolic pathway for bile salts(Fig. 10). Implications pertaining to detoxificationbecame immediately apparent. Lithocholate, being amonohydroxylated bile salt, is less water-solubleand more lipid-soluble than substances with two orthree hydroxyl groups. As a result, one observessignificant passive diffusion across the intestine.Figure 11 shows results of the in vivo intestinal ab-sorption studies of these compounds, done withguinea pigs (32). Distal and proximal segments wereperfused and transmucosal absorption was moni-tored by following the recovery of the test substratein the bile. On the left of Figure 11 we see thatconsiderable absorption of taurolithocholate andglycolithocholate can be observed from proximal aswell as distal regions, suggesting that both passive

Time in Minutes

FIGURE 11. Absorption of taurolithocholate and glycolithocholate(left) and of their 3-a-sulfate esters (right) as measured byrecovery of radioactivity from a bile fistula after in vivo perfu-sion of proximal and distal segments of small intestine from(32).

December 1979

Bllc

87

process and active transport systems can operate inabsorbing these substances. Following sulfation,one can detect absorption only from the distal regionof the small bowel suggesting that the passive flux ofthese three sulfate derivatives is minimal. Further-more, ileal absorption of the sulfated compoundcould be inhibited when they were perfused togetherwith primary bile salts. These animal studies sug-gested that the hepatic sulfation in the case of thelithocholates might be an adaptive mechanism toenhance their fecal excretion. Subsequently, Hof-mann et al. observed essentially the same pattern inhuman studies (33).

It must be noted that these sulfate esters bear twonegative charges at physiological pH. We havestated earlier that much of our ideas concerningcarrier-substrate interactions rests on the observa-tion that bile salts modified to have two negativecharges on the side chain appeared not to be trans-ported. Here when the second negative charge wasintroduced at the other end of the molecule there wasobviously some transport. In vitro studies confirmthese in vivo observations. Although the 3-position isdisplaced from the side-chain region, we were stillnot completely comfortable with our conclusionsconcerning two charges on the molecule. Therefore,it was important to ascertain whether sulfate es-terification had a quantitative effect on substratetmnsport. Unfortunately, the properties of tauro-lithocholate and glycolithocholate were such as toprevent a quantitative evaluation of the effect ofsulfation at this position. As demonstrated, conju-gated lithocholates being monohydroxylated bilesalts can to a great extent cross the intestinal mucosa

by passive means. In addition, these substanceshave a strong tendency to bind to tissue in a non-specific manner. Therefore, in vivo and in vitroevaluations of the ileal transport of lithocholic acidconjugates could not be accurately assessed. Thesecritical complications of passive fluxes and non-specific tissue binding do not pertain to the naturallyoccurring di- and trihydroxylated bile salts. There-fore, when it was reported that sulfation of di- andtrihydroxylated bile salts can take place, and thatthese processes are enhanced in patients withhepatobiliary disease (34-37), we decided to reinves-tigate this question. The primary bile salt,taurochenodeoxycholate, was selected because itallows for the assessment of the effect of sulfation atthe 3-position, the 7-position, and the 3,7-position.These bile salt esters were prepared and tested for,their ability to be absorbed by the intestine (38). Invivo perfusion of segments of small bowel withlabeled sulfate esters showed that sulfation markedlydecreased transport by the ileal bile salt transportsystem and that the position and number of the sul-fate radicals was directly correlated with the degreeof transport inhibition. The following structure re-lationships were found: transport of taurocheno-deoxycholate (TCDC) > TCDC-3-sulfate > TCDC-3,7-disulfate with a decrease in magnitude of ap-proximately 90%1o between each pair (Table 2). Sulfa-tion thus can be envisioned as a means of enhancingexcretion by the fecal route in conditions of partialobstruction. It would also appear that ileal transportof the monosulfates is less when the sulfate ester iscloser to the side chain.Enhanced renal excretion of sulfated bile salts has

Table 2. Intestinal absorption of sulfated derivatives of taurochenodeoxycholatefrom distal and proximal regions of the small bowel of guinea pigs.

Proximal segments Distal segments

No. of Absorption, No. of Absorption,Substrate animals nmole/g dry wt. animals nmole/g dry wt.

Taurochenodeoxycholate 4 73.2 ± 7.2 4 2290.0 ± 160.2aTaurochenodeoxycholate-

3-sulfate 4 4.7 ± 1.7 5 203.0 24bTaurochenodeoxycholate-

7-sulfate 5 3.1 ± 0.8 5 25.6 4.2cTaurochenodeoxycholate-

3,7-disulfate 4 4.8 ± 1.8 4 3.4 0.9d

abcI'hese values for ileal absorption are significantly different from each other: a and b,p<0.0001; b and c,p<0.0001; c and d,p<0.001by Student's t-test. In the case of the experiments with taurochenodeoxycholate (TCDC), TCDC-3-sulfate, and TCDC-7-sulfate, distalabsorption was significantly greater than the respective proximal absorption: p < 0.001 in all cases. Distal and proximal absorption ofthe TCDC-3,7-disulfate were not significantly different from each other.Perfusion solutions consisted ofnormal saline buffered with 0.01M Na phosphate, pH 6.9 with bile salts added in a concentration of 32nmole/ml. The rate ofperfusion was 3.5 mlmin. Perfusion with labeled bile salt was performed fora standard period of60 min. This waspreceded by a flush with buffered normal saline for at least 10 min and was followed by perfusion with buffered normal saline until nofurther radioactivity appeared in the bile (38).

Environmental Health Perspectives88

been demonstrated by patients with biliary obstruc-tion. Its relevance to the problems under considera-tion stems in part from an early observation of oursthat a similar transport system (for bile salts) exists inthe renal tubule which operates in the reabsorptivedirection (39). If it has the same structure-activitycharacteristics as the ileal transport system, then thebile salts following sulfation would not be reab-sorbed as readily as their unsulfated progenitors andas such be more likely to be excreted in the urine.

The author's work reported here was supported by GrantAM09582 from the National Institute of Health.

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