PROGRESS IN GASTROENTEROLOGY

Transcription

1 GASTROENTEROLOGY Copyright 1972 by The Willia ms & Wilkins Co. Vol. 62, No.1 Printed in U.S.A. PROGRESS IN GASTROENTEROLOGY THE ENTEROHEPATIC CIRCULATION R. HERMON DOWLING, M.D., M.R.e.p. Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, England Any substance secreted in bile which is reabsorbed from the intestine and returns to the liver to appear once again in bile may be said to undergo an enterohepatic circulation (EHe). Accepting this definition, the list of endogenous and exogenous substances with an EHe is formidable. The lipids in bile, cholesterol, phospholipids, and bile salts, provide examples of endogenous substances which all undergo enterohepatic circulations but with widely differing efficiencies (assuming that 100% absorption and reexcretion is the most efficient EHe). Of these biliary lipids, the EHe of bile salts is the most important but since individual molecules of both cholesterol and lecithin, the principal phospholipid in bile, may also circulate from bile to intestine and back to the liver and bile, they may be considered as having an EHe. Based on studies with labeled cholesterol, an EHe for cholesterol was postulated in and later by other authors. 2-~ Endogenous biliary cholesterol is probably mainly absorbed in the jejunum 5 and of this reabsorbed fraction, a small percentage may reappear in bile having followed the circuitous route of lymphatic transport, systemic circulation, hepatic artery, liver, ~md bile. However, there are many intermediate pools in which the individual cholesterol molecule may become sidetracked on the way and cholesterol, therefore, does not undergo Received July 27, Address requests for reprints to: Dr. R. Hermon Dowling, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12, England. 122 the "bulk" enterohepatic circulation of bile salts. Phospholipids, the other major lipid class in bile, resemble cholesterol in that a small percentage of biliary lecithin, which passes into the intestine, may find its way back to the bile. However, it differs from cholesterol in that it is digested in the intestinal lumen by pancreatic phospholipase to lysolecithin, and after absorption, is reesterified by the intestinal epithelium. A small percentage may find its way back to the bile. However, Saunders6 has recently shown convincingly that, quantitatively, the EHe of phospholipids is insignificant. Other endogenous substances with an EHe include bile pigments,7 vitamin B 12, a small percentage of which recycles each day, 8 folic acid,9, 10 and many estrogenic sterols. II, 12 Examples of ex, ~enous substances which undergo an EHe include many drugs such as flufenamic acid,13 digitalis glycosides, 14 and glutethimide,15 although the concept of an EHe for this last drug has recently been challenged. 16 This review discusses the normal physiology of the EHe of bile salts both in experimental animals and in man. (Different aspects of the bile salt EHe have been reviewed recently in this journap7. 18 and elsewhere ) The applied physiology of an interrupted EHe is discussed in detail since the altered bile salt metabolism of patients with ileal dysfunction may play a major role in the pathogenesis of diarrhea, renal calculi, and gallstone formation, from which ileectomy patients may

2 January 1972 PROGRESS IN GASTROENTEROLOGY 123 suffer. However, the role played by bile salts in regulating synthesis and secretion of biliary cholesterol and phospholipids is only briefly mentioned. It is now well established that the body conserves the bile acids by reabsorption from the intestine and that the bulk of this reabsorption is by active transport from the ileum. However, it is also known that other parts of the intestine, namely the stomach,22 jejunum,23-26 and colon may absorb bile acids by passive processes. The normal bile salt EHC in man may be summarized as follows. A 3- to 5-g bile salt pool consisting of both primary bile salts (synthesized in the liver from cholesterol) and secondary bile salts (resulting from bacterial conversion of primary bile acids in the intestine) circulates from liver to bile (with intermittent storage and concentration in the gallbladder), to intestine, to portal blood, and back to liver about six to 10 times per day.3 2, 33 Between 2 and 5% of the total circulating bile salts (or about 20 to 25% of the total bile salt pool34) escape reabsorption to appear in the feces giving a fecal bile salt excretion of approximately 200 to 600 mg per day In the steady state, since synthesis must equal loss, hepatic bile salt synthesis is therefore 200 to 600 mg per day. Quantitatively, fecal excretion is normally the only route for bile salt loss from the EHC (about 2% of the total daily bile salt loss is accounted for by urinary and skin excretion 38). It has been estimated (in the rat) that at anyone time about 85% of the circulating bile salts are present in the intestinal lumen, about 10 to 12% in the gut wall, and only 3 to 5% in the liver. 27 Function of Bile Salts in the Intestinal Lumen The EHC of bile salts ensures an economic conservation of these "biological detergents." Furthermore, the localization of the major transport site to the ileum presumably means that once the bile salt micelle has delivered its complement of lipolytic products to the small bowel epithelium, it may be reutilized to solubilize other lipids. If this concept is correct, then as chyme is propelled along the intestine, the bile salts provide a shuttle service between the oil water interface of emulsified fat droplets where hydrolysis of dietary fat by pancreatic lipase takes place, and the intestinal epithelium of the upper small bowel, where fat absorption is thought to occur.32, 39 Th~ simple concept of bile salts shuttling between emulsified fat in the bowel lumen and the microvillous membrane is still unproven, since at present it is not known exactly how monoglyceride and fatty acid molecules are absorbed from the micelle at the brush border of the absorptive epithelium. It seems likely, however, that fat molecules passively diffuse across the membrane, thereby disturbing the thermodynamic equilibrium of the micelle and creating vacancies for further lipid molecules to be solubilized. Hepatic Component of the Bile Salt EHC Quite apart from bile salt synthesis, the liver has two other roles in bile salt metabolism. It both conjugates any free bile salts which are delivered to it and it "clears" or secretes the bile salts into the bile by an active mechanism. This clearance is extremely efficient. In the dog, O'Maille et al. 40 found that 92% of labeled taurocholate given intravenously was cleared in one passage through the liver. The glycine conjugate, glycocholate, which is not a naturally occurring bile salt in the dog, was cleared equally rapidly. Bile salt conjugation. Normally there are no free or deconjugated bile salts in bile. Both newly synthesized and presumably also recycled bile acids, which have been deconjugated by intestinal bacteria before their reabsorption, must therefore be conjugated with the amino acids glycine or taurine before they are secreted in bile. To take cholic acid as an example, in the presence of adenosine triphosphate, the free bile acid is activated by coenzyme A to form cholyl CoA which, in turn, combines with the amino acid glycine to form glycocholate (or in the case of taurine, to form taurocholate). This conjugation

3 124 PROGRESS IN GASTROENTEROLOGY Vol. 62, No.1 step-peptide linkage at the carboxyl group of the bile acid-must also be efficient since when O'Maille and colleagues 4o injected free cholic acid, rather than its glycine conjugate, intravenously, 79% was cleared by the liver in one circulation. Normally, there are two factors which regulate the choice of amino acid for bile salt conjugation 41, 42-first, a species difference, and second, the availability of the sulfur-containing amino acid taurine or its precursors cystine, cysteine, and methionine. Considering the species difference, bile acids in the rat are almost exclusively conjugated with taurine while in the rabbit glycine conjugates predominate. Bremer43 showed that the hepatic microsomes of the rat selectively conjugated bile acids with taurine while those of the rabbit used glycine. Although in man the glycine conjugates normally predominate, the glycine to taurine (G: T) ratio being approximately 2 or 3: 1,44-48 the human liver conjugates bile acids preferentially with taurine. 49 In fact, after feeding taurine in doses from 3 to 15 g per day for 5 days, Sjova1l 44 showed that the normal G: T ratio became inverted so that 96% of the bile acids present became conjugated with taurine. The usual dietary intake of taurine or its precursors in man is such that it is normally in relatively short supply. In contrast, glycine, which is involved in many metabolic pathways, is freely available. Conjugation with bile acids is the only specific route for taurine catabolism. The factors regulating the choice of amino acid for bile acid conjugation are particularly important when the enterohepatic circulation is interrupted-for example by ileal dysfunction. Whether a bile acid is conjugated with glycine or taurine will influence its physical state in the gut and hence its rate of reabsorption from areas of the intestine other than the ileum. The question of which amino acid is used for conjugation may also be important in the genesis of hyper oxaluria and urinary oxalate stone formation. 50, 51 This, and other aspects of the broken EHC are discussed later. Choleretic effect of bile salts. The active transport of bile salts by the liver is the major, but not the sole, factor regulating the volume of bile secreted. 52 At zero bile salt secretion, the liver still secretes a watery bile-the so-called "bile salt-independent fraction."53 The concept of a bile salt-independent fraction was derived from studies where bile volume was plotted on the ordinate with bile salt secretion on the abcissa. When this is done, a straight line is found and by extrapolation this line does not go through the origin but intersects the ordinate. The bile salt-independent fraction does not apply to all species, since in the dog the linear relationship between bile volume and bile salt excretion passes close to zero. 55 In much the same way, there appears to be a relationship between the secretion rates for bile salts and phospholipids and those for bile salts and cholesterol in bile. The intimate relationship between bile salts, phospholipids, and cholesterol has been shown in isolated perfused livers of rats and dogs 60 and by bile fistula techniques in monkeys 61 and in man. 62 In fact, Nilsson and Schersten63 have shown, by in vitro studies using human liver slices, that bile acids regulate phospholipid synthesis. Hepatic bile acid synthesis and its control. The steps involved in hepatic bile acid synthesis and the way in which this synthesis is regulated have been the subject of recent studies by Shefer et al The liver synthesizes both cholesterol and bile acids-cholesterol being synthesized from acetate along the hydroxymethylglutarate CoA, mevalonate, cholesterol pathway. In turn, cholesterol is catabolized to 7 a-hydroxy cholesterol and bile acids. A detailed account of this complex pathway is beyond the scope of this review, but briefly, an a-oh group is added in the 7 position, the spatial orientation of the OH group in the 3 position (common to all primary mammalian bile acids) changes from (3 to a. The Ll5 double bond of the cholesterol molecule is reduced, bile acids being fully saturated, and in the case of cholic acid, a further OH group is added in

4 January 1972 PROGRESS IN GASTROENTEROLOGY 125 the 12 position before the side chain of the 27-carbon atom cholesterol molecule is shortened by 3 carbon atoms. 66 Hepatic bile acid synthesis is regulated by feedback inhibition exerted by the amount of bile acids returning to the liver. This was shown by Bergstrom and Danielsson67 who reinfused bile acids into the distal end of the common bile duct in the bile fistula rat. By so doing, they were able to inhibit the rebound or compensatory increase in synthesis which is seen after the EHC is broken by fistula diversion of bile. These studies were confirmed by She fer et al.,64 who returned bile salts to the EHC by duodenal infusion and quantitated the amount of bile acid needed to inhibit the rebound increase in synthesis. They then extended these studies to define, with labeled precursors, at which step in the acetate to bile acid pathway, the regulation of bile acid synthesis took place. They concluded that 7a-hydroxy cholesterol was the rate-limiting step, and that 7a-hydroxylase was the rate-limiting enzyme. This confirms earlier studies by Danielsson et al. 68 Danielsson 69 has suggested that the major pathway for cholic acid formation from cholesterol is by this 7a hydroxylation step although the bile acid may also follow a less important pathway through 26 OH cholesterol. 70 The enzyme cholesterol 7a-hydroxylase is the first unique enzyme in bile acid biosynthesis and in recent years it has been extensively studied. It may be increased (up to 6-fold) when the EHC is broken with a bile fistula 68 or after feeding cholestyramine. 71 The enzyme is produced by the endoplasmic reticulum 71 but, unlike other microsomal enzymes such as cytochrome P450, Boyd et al. 71 found that it was not significantly induced by phenobarbitone. However, it has been suggested recently that small doses of phenobarbitone in the primate do significantly increase bile salt synthesis.72 Furthermore, this key enzyme in bile acid synthesis is believed to undergo a circadian rhythm with maximal acitivity in late afternoon. This diurnal variation is, in part at least, under hormonal controp3 since it is inhibited by hypophysectomy and/or adrenalectomy.74 The factors regulating the secretion of bile and the passage of bile acids from liver and gallbladder into the intestine are complex. Food is of major importance in stimulating both bile volume and bile salt secretion presumably because, together with gastric acid, it stimulates cholecystokinin, glucagon, and secretin release from duodenal mucosa. In turn, these polypeptide hormones stimulate the liver to secrete a watery bile rich in bicarbonate and in the case of cholecystokinin, provoke gallbladder contraction. Gastrin, like secretin and glucagon, stimulates the hepatocytes to secrete a bicarbonate rich watery bile Intestinal Bile Acid Absorption Intestinal bile acid transport may be summarized as follows: (1) Active transport from ileum alone, which is the major route for bile salt reabsorption. (2) Passive absorption from stomach, jejunum, ileum, and colon. (a) passive monomer diffusiondiffusion of individual bile salt molecules when the intraluminal bile salt concentration is below the critical micellar concentration (rare) or diffusion of single molecules which always coexist in equilibrium with the molecular aggregates of bile salt molecules-the micelles. (b) passive micellar diffusion. These passive processes may be in the ionic or non ionic form but since the transport of charged particles is inhibited by the electrical gradient in the intestine, nonionic diffusion is more important. Active Ileal Transport The active transport of bile salts in the ileum has been repeatedly demonstrated in the last decade, 18, 20, since Baker and Searle93 confirmed earlier studies by von Tappeiner94 by showing in vivo, that bile acids were absorbed from the ileum in the rat. The localization of active bile salt transport to the ileum has also been shown for hamsters, rabbits, 95, 96 dogs89, 95 and many other species includ-

5 126 PROGRESS IN GASTROENTEROLOGY Vol. 62, No. 1 ing Leghorn chickens, King pigeons, mice, squirrel monkeys, and spider monkeys. 95 The comparative efficiency of active ileal transport for different bile acids was reported by Lack and Weiner,90 who found that the dihydroxy bile acids (deoxy- and chenodeoxycholic acid) were absorbed less efficiently than the trihydroxy bile acid (cholic acid) and that the taurine conjugates were transported more rapidly than the glycine conjugates. Furthermore, Heaton and Lack97 showed that there was mutual inhibition of transport of glycine and taurine conjugates. The quantitative significance of these findings in terms of the EHC in man has yet to be evaluated. Passive Absorption Influence of physical properties on bile acid absorption. Passive bile acid diffusion may be either from the ionized or from the nonionized form, which in turn depends on the prevailing ph and the dissociation constant (pka) of the individual bile acid. Thus, at normal intestinal ph levels of 5.0 to ,103 a greater proportion of free bile acids with pka's of 5.0 to 6.3 will be in the protonated (or nonionized) form than the glycine-conjugated bile acids (pka's 4.3 to 5.2) while the stronger taurine-conjugated bile acids (pka's 1.8 to 1.9) are almost entirely in the ionized form. 104 Since passive bile acid absorption by non ionic diffusion is at least 5 to 6 times greater than diffusion of charged particles,24 free bile acids are absorbed more rapidly from extra-ileal sites than glycine conjugates while the ionized taurine conjugates are almost totally dependent on the active transport site in the ileum for their reabsorption. 25 The physical properties of the different types of bile acids are therefore of considerable importance in determining not only their rates of absorption, but also their own solubility in the intestinal lumen 104 and their capacity to solubilize other lipids in the small bowel. However, bile acids do not have fixed critical micellar concentrations (CMC's), dissociation constants (pka's), and lipid-solubilizing capacities. The CMC of an individual bile acid in vitro is not constant but is related to ph, temperature, and counter ion concentration. 105, 106 The pka is related to bile acid concentration 107 and perhaps to a limited extent to counter ion concentration while the ability of a given bile acid to solubilize cholesterol, for example, is greatly altered by the presence of other lipids such as phospholipids which expand the micelle, 105, 109 thereby enhancing its lipid-solubilizing capacity.105, Furthermore, the CMC of a bile acid depends to a certain extent on the method used to measure it There is, therefore, no simple magical figure for a bile salt CMC below which concentration lipolytic products are not solubilized and above which fat solubilization and absorption proceeds satisfactorily. In general, however, once the CMC is exceeded, the greater the bile salt concentration, the greater is the amount of fat solubilized. This concept is illustrated by the findings of Badley et ai., 113 who showed that in patients with chronic liver disease, those with steatorrhea had intraluminal bile salt concentrations of less than 4 J1moles per ml while those with normal fecal fat excretion had bile salt concentrations exceeding 6 J1moles per ml-a concentration found in control subjects and comparable to other reported normal values , 103, 114, 115 Based on their studies of intraluminal bile salt concentrations in liver disease, Badley et al. 116 suggested that the term "critical physiological concentration" rather than CMC was more relevant in clinical investigation. The relationship between physicochemical properties of bile salts and their function has been fully reviewed elsewhere. 19, 105 Quantitative significance of extra-ileal bile salt absorption. The quantitative significance of passive absorption from extraileal sites is controversial. Although bile salts can be absorbed at a particular rate by passive diffusion, this does not mean that they are necessarily absorbed at that rate under physiological conditions. Conversely, although active bile acid transport is confined to the ileum, this does not mean that the distal small bowel is the sole site for bile salt transport. While the role of passive extra-ileal bile salt absorption may

6 January 1972 PROGRESS IN GASTROENTEROLOGY 127 be less important for the normal EHC, it assumes greater significance in clinical situations where EHC is broken. For example, extra-ileal bile salt absorption is of considerable importance in ileal dysfunction, following cholestyramine, lignin, or neomycin treatment where bile acids are complexed or precipitated in the intestinal lumen, and in the blind loop syndrome when bacterial deconjugation renders bile acids vulnerable to premature absorption by diffusion 25 or perhaps to precipitation at normal intestinal ph, as was postulated recently on the basis of in vitro titration studies. 104 Bile acid precipitation may certainly occur in patients with intestinal stasis since bile acid enteroliths have been found in patients with jejunal diverticulosisl and in other examples of the blind loop syndrome However, the quantitative significance of bile acid precipitation as a mechanism for conjugated bile salt deficiency in the blind loop syndrome has yet to be established. Bile Acid Transport to the Liver Following their absorption, bile acids are transported to the liver in the portal veinl bound to albumin and to a lesser extent to other proteins In 1955, Sjovall and Akesson 125 showed that although oral 14C-taurocholate could be recovered in the bile, none was found in lymph, suggesting that it was absorbed by another route-presumably by the portal vein. More recently, Reinke and Wilson 129 showed that although the concentration of bile acid carrier protein was only twice as high in lymph as in blood, portal blood flow was 280 times that of lymph. The difference in flow rates, therefore, is the principal factor governing bile acid transport by the portal route. In the rat, as much as 36% of bile acids in the portal blood may be in the free form.130 However, the rat is coprophagic and much higher concentrations of bacteria capable of deconjugating bile salts are found in the normal rat jejunum when compared with man. It is unlikely, therefore, that such a high percentage of unconjugated bile salts would be found in human portal blood. Serum Bile Acids In man, the concentration of individual bile acids in the systemic circulation is normally very low, the total serum bile acids ranging from 2 to 4 Jlmoles per liter131-about one-thousandth that found in the intestinal lumen. In serum, bile acids are normally in the conjugated form, but in intestinal diseases such as the blind loop syndrome and in patients with intestinal resection, there are elevated concentrations of total bile acids in the systemic circulation, largely due to the presence of free bile acids. 132 Since free bile acids are cleared by the liver almost as efficiently as are the conjugated forms, the mechanism for the raised levels of serum free bile acids in conditions of intestinal stasis is not clear. The explanation may be that free bile acids are more avidly bound to albumin in the serum with a resultant differential hepatic clearance rate between the bound free and the unbound conjugated biles acids. 128 High levels of bile acids in the serum of patients with obstructive jaundice, intrahepatic cholestasis, pruritis, and jaundice of pregnancy have been known for some time However, in this situation the mechanism for high serum bile acid concentrations is different from that in the blind loop syndrome since in the cholestatic disorders there is impaired hepatic uptake, the mean normal half-life of intravenously 14C-cholic acid of 12.6 min increasing to 45_ min. 133 While difficult to document accurately for technical reasons, there are also increased quantities of bile acids in the skin of patients with obstructive jaundice 137 which are thought to cause the pruritis by irritation of sensory nerve endings. A rough correlation was found between the severity of itching and the quantity of bile acid recovered from the skin. 137 Why high levels of serum bile acids in patients with the blind loop syndrome or ileal resection should not cause itching similar to that seen in obstructive jaundice is not clear. It may be that only conjugated bile acids are capable of causing itching as opposed to the free form which is found in the serum of the blind loop patient.

7 128 PROGRESS IN GASTROENTEROLOGY Vol. 62, No.1 Role of Bacteria The principal actions of intestinal bacteria on bile salts may be summarized as follows: (1) deconjugation-removing taurine and glycine from the conjugated bile salts; (2) 7a dehydroxylation-removing the OH group from the 7 position of the primary bile salts, cholate and chenodeoxycholate, producing deoxycholate and lithocholate respectively; (3) oxidation of the hydroxyl groups to produce keto bile acids; (4) epimerization with changes in the spatial orientation of a-hydroxyl groups to the f3 position. The liver in man synthesizes two primary bile acids, the trihydroxy cholic acid and the dihydroxy chenodeoxycholic acid. However, analysis of human bile shows that roughly one-quarter to one-third of the total bile acids are secondary bile acids-deoxycholic acid and occasionally trace amounts of lithocholic acid. These secondary bile acids are the result of bacterial enzymatic 7 a dehydroxylation of the primary bile acids. Thus, removal of the hydroxyl group from the 7 position of cholic acid produces the secondary dihydroxy bile acid deoxycholic acid while lithocholic acid (a monohydroxy bile acid) is formed by 7 a dehydroxylation of chenodeoxycholic acid. When the primary bile salts are prevented from coming in contact with intestinal bacteria, for example, by diversion of bile with a fistula 138 or when the common bile duct is occluded by a stone, 139 deoxycholate rapidly disappears from the circulation. Norman 140 has shown in vitro that before bacteria remove the hydroxyl (OH) group from the 7 position, deconjugation must first occur. The deconjugating enzyme seems to be widely distributed among anaerobic intestinal bacteria such as Bacteroides, Veillonella, Bifodobacteria (anaerobic lactobacilli) and some strains of clostridiae, 142, 145 but dehydroxylating enzymes seem much less common. 140 Sites of secondary bile acid formation and absorption. The concentrations of anaerobic organisms increase to concentrations of 107 to 109 per ml in the distal ileum-concentrations similar to. those found in the colon and in feces. 146, 147 It is likely, therefore, that deoxycholate and lithocholate are formed in the lower ileum as well as in the colon. Lithocholate is extremely insoluble and has a very high critical micellar temperature (CMT or "Krafft" point).148 Because of these physical properties, it does not form micelles at body temperature, is poorly absorbed, and as a result, it is quantitatively unimportant in the normal EHC of bile salts. Where deoxycholate is absorbed to enter the EHC is largely an open question, although available evidence suggests that it is absorbed from the colon. Again, although deoxycholate can be absorbed from the cecum and colon, this does not necessarily mean that this is its normal site of absorption. Nor would studies with ileal intubation answer the question, since intraluminal deoxycholate concentrations at this site are the net result of both rates of formation and rates of absorption. However, the limited available evidence suggests that although deconjugation may occur normally in the ileum, 149 dehydroxylation of the liberated cholate does not occur until the large bowel. 150 In patients with ileostomy following proctocolectomy for ulcerative colitis, the ileal effluent contains high concentrations of bacteria-los to 108 per ml 143 capable of salt deconjugation 142, 151 and in fact, free bile salts may be demonstrated in the efeffluent.143 Although many of these same bacteria are also capable of dehydroxylation in vitro, no deoxycholate is present either in ileal effluent or in bile. If the findings in ileostomy patients may be extrapolated to the normal EHC, deoxycholate must be passively reabsorbed from the colon. The secondary bile acids produced by bacterial action, deoxycholate and lithocholate, make up the bulk of bile acids excreted in the feces. 35, 36 However, in addition to deconjugation and dehydroxylation, bacteria can also oxidize the hydroxyl groups to yield keto groups and other metabolites. In fact, about 19 differ-

8 January 1972 PROGRESS IN GASTROENTEROLOGY 129 ent types of bile acid have been identified in human feces. 35, 152 Effects of Interruption of the EHe Studies on the effects of interruption of the EHC have been among the most interesting developments of applied gastrointestinal physiology in recent years. The EHC may be interrupted either when bile salts are prevented from reaching the intestine, as in obstructive jaundice, or when intestinal absorption is blocked by intraluminal events such as bile acid binding by drugs-for example cholestyramine l53, 154 and lignin,155 or by bile acid precipitation as in the Zollinger Ellison syndrome 156 or following neomycin treatmentt l5, 157, 158 and perhaps also in the blind loop syndrome. 104 Intestinal malabsorption may also interrupt the EHC, for example in patients with resection, bypass, or disease of the ileum (including Crohn's disease,159 celiac disease, and tropical sprue 162). Ileal disease, resection, and bypass. Since the ileum is the major site for bile salt reabsorption, ileal disease or resection should cause bile salt malabsorption, and in 1965 this was clearly demonstrated in dog. 163 These authors fed isotopic taurocholate to animals with a cannula in the gallbladder and showed that the percentage of recovery oflabeled material was similar in controls and in dogs with proximal resection (48%), but only 3% was recovered from ileectomized dogs. Since then, increased fecal bile salt excretion and/or markedly shortened half-lives of isotopic bile salts have been repeatedly demonstrated both in experimental animals and in man. 29, 47, 48, 159, As a result, many, but not all, of these patients have intraluminal bile salt deficiency in the jejunal lumen_ , The bile salt concentrations in the jejunal lumen of ileectomized patients may vary throughout the day_ It has been suggested that, during an overnight fast of about 12 hr, the maximal rates of hepatic bile salt synthesis may reconstitute the bile salt pool which, in the absence of foodstimulated cholecystokinin release, is stored in the gallbladder. In response to breakfast, therefore, the gallbladder contracts to produce normal jejunal bile salt concentrations. In the absence of an intact ileum, however, the bile salt pool is malabsorbed on the first enterohepatic cycle and even with maximal synthesis, in the relatively limited period of time between meals, the bile salt pool cannot be restored. As a result, the intraluminal bile salt concentrations are depleted after the mid-day and evening meals. 169 This diurnal variation in jejunal bile salt concentration after ileal resection is illustrated by the results of a study in our laboratory (R. H. Dowling and C. B. Campbell, 1971, unpublished observations) (fig. 1). After an overnight fast, in a patient with a massive distal small bowel resection, jejunal bile salt concentrations in response to a test meal were normal and the amount 9.00 A. M. (Fasti ng) 3. OOP. M. I Fed) INTRA- 1Jl moles LUM INAL 1m!. concentration Range.. BI LE ~ I SALTS m moles DISTR IBUTION Of INTRA-LUMINAL 60 FAT FIG. 1. Diurnal variation in intraluminal bile salt concentrations after ileal resection. Jejunal bile salt concentrations and phase distribution of fat aspirated from the intestinal lumen over three half-hour periods, following a test meal in a patient with a massive distal small bowel resection (14 inches jejunum remaining, anastomosed to midtransverse colon). The study was performed initially after an overnight fast and repeated 1 week later in midafternoon (see text). (R. H. Dowling and C. B. Campbell, unpublished observations).

9 130 PROGRESS IN GASTROENTEROLOGY Vol. 62, No. 1 of fat in the micellar phase of ultracentrifuged intestinal aspirates was also normal. I15 However, on a second occasion 1 week later, when the study was repeated after breakfast, mid-morning snack, and lunch, there was intraluminal bile salt deficiency with reduced fat in the micellar phase and a corresponding expansion of the oil phase. Furthermore, the total amount of bile salt aspirated during the three 1/2-hr periods of the study (a crude measure of the bile salt pool) was normal during the early morning study, but markedly diminished at 3:00 PM. The observation that ileal resection may produce jejunal bile salt deficiency provided one possible explanation for the apparent paradox that while fat absorption normally occurs in the jejunum, removal of the ileum produces a greater degree of malabsorption than does resection of a comparable length of jejunum. 173 Other factors such as the adaptive ability of the ileum to increase its absorptive capacity and differential rates of transit through jejunum and ileum also account for the paradoxical steatorrhea after ileectomy. 174, 175 The bile salt deficit which follows ileal resection leads to impaired micelle formation and reduced absorption of fat and particularly of sterols such as cholesterol and the fat soluble vitamins whose absorption is dependent on bile salts to a much greater extent than dietary triglycerides, which may be absorbed even in the absence of bile salts. 176, 177 In patients with ileal dysfunction, there sometimes is a discrepancy between bile salt malabsorption with shortened halflives of isotopic bile salts, and normal concentrations of bile salts in the jejunum. In the absence of a normal ileum, the jejunal bile salt concentration depends, on one hand, on increased bile salt loss in the feces due to malabsorption, and, on the other hand, on the ability of extra-ileal sites to partially maintain the EHC, coupled with the adaptive increase in hepatic bile salt synthesis. Compensatory increase in hepatic bile salt synthesis. The precedent that the liver is capable of a compensatory increase in bile salt synthesis after ileal resection is well established from studies where there has been a broken EHC from other causes. After creating a bile fistula in rats, for exam pie, Eriksson 178 found a 10- to 20- fold increase in hepatic bile salt synthesis, which was later confirmed by others,64, , 179, 180 although in Myant and Eder's experiments, 181 there was only a 4- to 5-fold increase in synthesis. Similar increases in hepatic bile salt synthesis have been found after feeding cholestyramine to mice l82 and to man However, it should be stressed that synthesis normally contributes such a small fraction to the total circulating bile salts, that even a 10- to 20-fold increase in synthesis is usually totally inadequate to compensate for the increased fecal loss which follows ileal resection. Recently, detailed studies of the adaptive increase in hepatic bile salt synthesis were made in the rhesus monkey, comparing the long term effects of mechanical interruption of the EHC with varying degrees of ileal resection. 172 Bile from a chronic bile fistula was returned to the intestine through an electronic stream splitter, which diverted varying percentages of bile to the exterior, thus providing controlled interruption of the EHC.57 The rhesus monkey can only compensate for 20% interruption of the EHC by a 4- to 5-fold increase III hepatic bile salt synthesis. Assuming that steady state kinetics exist in patients with ileal resection and that fecal bile salt excretion matches hepatic synthesis, a similar degree of compensation was calculated for patients with complete external biliary fistulae by Carey and Williams, 185 who estimated that the human liver could compensate for 16% interruption of the EHC. Extra-ileal bile 'salt absorption. The partial conservation of the EHC by bile salt diffusion from extra-ileal sites was suggested by studies in the rhesus monkey which showed that the more extensive the distal small bowel resection, the greater was the deficit in bile salt secretion. 172 Following ileal resection, the individual contributions by the residual jejunum and colon in bile acid reabsorption have not yet been documented. However, recent

10 January 1972 PROGRESS TN GASTROENTEROLOGY 131 studies from our laboratory18sa suggest that, per unit length, the jejunum is 2 to 3 times more effective than the colon in bile acid absorption. It also seems likely that a small adaptive increase in bile acid absorption occurs in the residual jejunum after removal of the ileum, comparable to the compensatory increase in glucose absorption seen after ileectomy,l74 although no such adaptive change is seen in the colon. Furthermore, following proximal small bowel resection, active bile salt transfer by the residual ileum becomes supranormal. 185a Bile salt markers for the broken EHC. The physiological markers of a broken EHC may be summarized as follows. Depending on the magnitude of bile salt loss due to ileal malabsorption, and the degree of success in restoring the EHC by the adaptive increase in hepatic bile salt synthesis and by extra-ileal bile salt reabsorption, there mayor may not be intraluminal bile salt deficiency. There is, however, increased fecal bile salt excretion and therefore increased hepatic bile salt synthesis. In turn, hepatic 7 a-hydroxylase activity is increased while 12ahydroxylase activity is suppressed. As a result, the ratio of chenodeoxycholate to cholate in both blood and bile is increased.186 Again, if cholic acid is prevented from contact with intestinal microorganisms, as occurs, for example, with a bile fistula,. the secondary bile acid, deoxycholic acid, disappears from the EHC and is no longer found in duodenal aspirates. 186, 187 Finally, the G:T bile salt ratio increases from the normal 2 or 3: to between 12 and 20:1. 47, 51,169, 188 There are two possible mechanisms for the increased G : T ratio. First, the increased hepatic bile salt synthesis rate rapidly drains the limited stores of taurine while the ubiquitous glycine is freely available. Second, in ileal dysfunction at least, there are theoretical reasons for postulating selective wasting "f taurine-conjugated bile acids. Because of their low pka's, at prevailing intestinal ph levels (5.0 to 7.0) the taurine conjugates are almost entirely in the ionized form and as such are unavailable for passive non ionic diffusion. In contrast, a considerable percentage of the glycine-conjugated bile acids are in the nonionized form at these ph levels and so their EHC is partially conserved by jejunal reabsorption. The quantitative contribution of each of these mechanisms is unknown, but depletion of taurine stores is probably the more important factor. Certainly the fractional catabolic rate of labeled glycocholate was the same as that of taurocholate when the turnover of these two isotopes was compared in patients with ileal dysfunction. 188 However, a comparable study with the glycine and taurine conjugates of chenodeoxycholic acid is needed, since the passive diffusion of dihydroxy bile salts is greater than that of cholic acid. 20 Clinical Consequences of the Broken EHC in Ileal Dysfunction Depletion of bile salts: steatorrhea and gallstone formation. The spillover of unabsorbed bile salts into the colon of patients with ileal dysfunction is thought to be an important factor in the pathogenesis of watery diarrhea from which many of these patients suffer. 189, 190 The resultant intraluminal bile salt deficiency contributes to the steatorrhea of the ileectomized patient. Furthermore, the depletion of biliary bile salts may jeopardize cholesterol solubility in bile, thereby predisposing cholesterol gallstone formation, Bile salt diarrhea in the broken EHC. The cathartic action of excessive amounts of bile salts in the colon, which results from ileal disease, has been emphasized recently.189, 190 Bile salts, particularly the dihydroxy bile salts, inhibit water, sodium, chloride, and bicarbonate absorption and promote potassium secretion The cathartic effect of excessive amounts of bile salts in the colon may also be related to accelerated motility, which has been shown in the dog,193 in the guinea pig,194 and in man. 19S Although patients with ileal dysfunction may have an intraluminal bile salt deficiency, replacement therapy with exogenous bile salts has not been successful in controlling their diarrhea. In fact, although fecal fat excretion may be improved, the diarrhea is usually aggravated,164, 166, 196

11 132 PROGRESS IN GASTROENTEROLOGY Vol. 62, No.1 presumably because malabsorption of exogenous bile salts simply compounds the cathartic or cholerheic load of endogenous material already spilling into the colon. 189 The apparent paradox of further reducing the available bile salts by feeding a bile acid-sequestering agent, cholestyramine l97 may in fact improve the patient's diarrhea Lignin, a derivative of wood pulp, also binds bile acids both in vitro, 155 and in the intestinal lumen. 202 It is said to selectively bind free bile acids and therefore theoretically its principal effect should be in the colon after bacteria have deconjugated the malabsorbed conjugates. If true, this would leave the conjugated bile salts free to promote micelle formation in the jejunum. 203 This hypothesis has not been supported in man,204 and lignin seems to have a similar but less effective bile acid-binding capacity than cholestyramine. 205 Its sole advantage, therefore, is probably that it is more palatable than cholestyramine, which, in spite of refinements in preparation, still has a somewhat fishy odor. Complications to treatment with cholestyramine have been recorded, however, including nausea, vomiting, constipation, and fecal impaction,154 acidosis due to increased bicarbonate loss in the stool,206 intestinal obstruction,207 and hemorrhage from hypoprothrombinemia.208 In spite of these rare complications, a therapeutic trial with cholestyramine is always worthwhile in an attempt to control the diarrhea of patients with ileal disease or resectionpreferably in the controlled environment of a metabolic unit. Hofmann and Poley l99 have suggested that patients with short lengths of ileum resected (less than 100 cm) who have watery diarrhea are the patients most likely to benefit from cholestyramine therapy, even at the expense of a modest increase in fecal fat. However, in the author's experience, response to treatment with this drug is variable and unpredicatable and is not dependent on the extent of the resection. Treatment of hypercholesterolemia by breaking the EHC. Since bile acid synthesis represents the major catabolic pathway for cholesterol, many attempts have been made to promote the excretion (and hence the synthesis) of bile acids in the management of hypercholesterolemia. The methods used have included ileal resection and bypass, and treatment with bile acid-sequestering (cholestyramine) and precipitating (neomycin) drugs. Although such treatment undoubtedly does increase both bile acid and neutral sterol excretion in the feces, unfortunately such interruption of the EHC also stimulates hepatic cholesterol synthesis and as a result, the initial lowering of serum cholesterollevels is not always maintained. 196 Oxalate metabolism in the broken EHC. An intriguing consequence of bile salt malabsorption and the attendant increase in G: T bile salt ratio is the occurrence of hyperoxaluria and renal calculi from which the patient with ileal disease or resection may suffer The proposed mech anism for the hyperoxaluria is as follows. Colonic bacteria deconjugate the malabsorbed glycine-conjugated bile acids and some of the liberated glycine is further converted by bacterial enzymes to glyoxalate which is absorbed, oxidized in the liver to oxalic acid, and excreted by the kidneys to produce hyperoxaluria. Since glycine is thought to be the substrate for this form of secondary hyperoxaluria, and since it has been known for some time that the G: T ratio may be reversed by feeding taurine,44 Dowling et al. 5 I fed taurine to one of their ileal resection patients and abolished the hyperoxaluria-a finding which has subsequently been confirmed. 217 Why some but not all patients with ileal dysfunction should develop hyperoxaluria is not at present clear. Radiorespirometry in the broken EHC and in the blind loop syndrome. Most isotopic bile salts studies use material labeled with 14C in the carboxyl position but l4c-glycine labeled glycocholate is now commercially available (Radiochemical Centre, Amersham, Bucks., England). Using this isotope, Hofmann et al. 50 showed that in patients with a broken EHC due to ileal dysfunction, in addition to the glycine -> glyoxalate -> oxalic acid pathway, some of the glycine may be converted to l4co 2' By trapping expired

12 January 1972 PROGRESS IN GASTROENTEROLOGY C0 2 in hyamine, Hofmann and his colleagues 50 were able to measure cumulative breath excretion of 14C0 2 with a liquid scintillation counting technique. Over a 12-hr period, 2 ileal resection patients excreted 31 and 36% of the isotope, compared with a mean of 8.4% (range 3.6 to 13.2%) in 5 normal controls. The bacterial catabolism of glycine may well be analogous to the formation of indican from dietary tryptophan. Raised levels of urinary indican excretion are found both in patients with the blind loop syndrome where excessive numbers of colonic bacteria attack normal dietary tryptophan in the small bowel, and also in malabsorption where the normal colonic flora attacks tryptophan which has spilled into the large bowel. 218 By the same analogy, the increased G: T ratio in p? tients with intestinal stasis 219 may provide the glycine substrate for oxalate formation and hyperoxaluria in the stagnant loop syndrome. This hypothesis has yet to be confirmed, but two recent studies have suggested that by monitoring expired radioactivity after oral HC-glycine labeled glycocholate, a marked increase in 14C02 production is found in the breath of patients with the blind loop syndrome The development of a simple breath monitoring test after an oral isotopic dose of glycine-labeled glycocholate to diagnose ileal dysfunction or intestinal stasis offers tempting possibilities. REFERENCES 1. Siperstein MD, Hernandez HH, Chaikoff IL: Enterohepatic circulation of carbon 4 of cholesterol. Am J Physiol 171 : , Stanley MM, Cheng SH: Cholesterol exchange in the gastrointestinal tract in normal and abnormal subjects. Gastroenterology 30:62-74, Borgstrom B: Quantification of cholesterol absorption in man by fecal analysis after the feeding of a single isotope-labeled meal. J Lipid Res 10: , Grundy SM, Ahrens EH, Davignon J : The interaction of cholesterol absorption and cholesterol synthesis in man. J Lipid Res 10: , Borgstrom B: Studies on intestinal cholesterol absorption in the human. J Clin Invest 39: , Saunders DR: Insignificance of the enterobiliary circulation of lecithin in man. Gastroenterology 59: , Lester R, Schumer W, Schmid R: Intestinal absorption of bile pigments. IV. Urobilinogen absorption in man. N Engl J Med 272: , Grasbeck R, Nyberg W, Reizenstein P: Biliary and fecal vitamin B 12 excretion in man. An isotope study. Proc Soc Exp Bioi Med 97: , Baker SJ, Kumar S, Swaminathan SP: Excretion of folic acid in bile. Lancet 1:685, Herbert V: Excretion offolic acid in bile. Lancet 1:913, Adlercreutz H, Schauman K-O: Excretion of oestrone and oestrior in the urine of some male subjects with the Dubin-Johnson syndrome and cirrhosis following oral administration of oestradiol benzoate. Acta Med Scand (suppl) 412: , Sandberg AA, Kirdani RY, Back N, et al: Biliary excretion and enterohepatic circulation of estrone and estriol in rodents. Am J Physiol 213: , Winder CV, Kaump DH, Glazko AJ, et al: Experimental observations on flufenamic, mefenamic, and meclofenamic acids, Pharmacology of the Fenamates. Ann Phys Med (suppl) 7-16, Caldwell JH, Greenberger NJ: Cholestyramine enhances digitalis excretion and protects against lethal intoxication (abstr). J Clin Invest 49:16a, De Myttenaere M, Schoenfeld L, Maher JF: Treatment of glutethimide poisoning. A comparison of forced diuresis and dialysis. JAMA 203: , Chary tan C: The enterohepatic circulation in glutethimide intoxication. Clin Pharmacol Ther 11 : , Hofmann AF: Clinical implications of physicochemical studies on bile salts. Gastroenterology 48: , Lack L, Weiner 1M: The role of the intestine during the enterohepatic circulation of bile salts. Gastroenterology 52: , Hofmann AF, Small DM: Detergent properties of bile salts: correlation with physiological function. Ann Rev Med 18: , Dietschy JM: Mechanisms for the intestinal absorption of bile acids. J Lipid Res 9: , Heaton KW: The importance of keeping bile salts in their place. Gut 10: , Davenport HW: Absorption of taurocholate-24- "c through the canine gastric mucosa. Proc Soc Exp Bioi Med 125: , 1967

13 134 PROGRESS IN GASTROENTEROLOGY Vol. 62, No Tidball CS: Intestinal and hepatic transport of cholate and organic dyes. Am J Physiol 206: , Dietschy JM, Salomon HS, Siperstein MD: Bile acid metabolism. I. Studies on the mechanisms of intestinal transport. J Clin Invest 45: , Hislop IG, Hofmann AF, Schoenfield LJ: Determinants of the rate and site of bile acid absorption in man (abstr). J Clin Invest 46:1070, Small NC, Dietschy JM: Characterization of the monomer and micelle components of the passive diffusion process of bile acids across the small intestine of the rat (abstr). Gastroenterology 54: 1272, Norman A, Sjovall J: On the transformation and enterohepatic circulation of cholic acid in the rat: bile acids and steroids 68. J Bioi Chern 233: , Portman OW: Further studies of the intestinal degradation products of cholic acid-24-c 14 in rats: Formation of deoxycholic acid. Arch Biochern Biophys 78: , Sullivan MF: Bile salt absorption in the irradiated rat. Am J Physiol 209: , Samuel P, Saypol GN, Meilman E, et al: Absorption of bile acids from the large bowel in man. J Clin Invest 47: , Gustafsson BE, Norman A: Bile acid absorption from the caecal contents of germ-free rats. Scand J Gastroenterol 4: , Borgstrom B, Dahlqvist A, Lundh G, et al: Studies on intestinal digestion and absorption in the human. J Clin Invest 36: , Lindstedt S: The turnover of cholic acid in man. Bile acids and steroids, 51. Acta Physiol Scand 40: 1-9, Bergstrom S: Metabolism of bile salts. Fed Proc 21 (suppl 11):28-32, Eneroth P, Gordon B, Ryhage R, et al: Identification of mono- and dihydroxy bile acids in human feces by gas-liquid chromatography and mass spectrometry. J Lipid Res 7: , Eneroth P, Gordon B, Sjovall J: Characterisation of trisubstituted cholanoic acids in human feces. J Lipid Res 7: , Evrard E, Janssen G: Gas-liquid chromatographic determination of human fecal bile acids. J Lipid Res 9: , Grundy SM, Ahrens EH, Miettinen TA: Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J Lipid Res 6: , Booth CC, Alldis D, Read AE: Studies on the site of fat absorption. 2. Fat balances after resection of varying amounts of the small intestine in man. Gut 2: , O'Maille ER, Richards TG, Short AH: The influence of conjugation of cholic acid on its uptake and secretion: Hepatic extraction of taurocholate and cholate in the dog. J Physiol (Lond) 189: , Schersten T: The synthesis of cholic acid conjugates in human liver. Acta Chir Scand (suppl) 373:1-38, Garbutt JT, Lack L, Tyor MP: Physiological basis of alterations in the relative conjugation of bile acids with glycine and taurine. Am J Clin Nutr 24: , Bremer J : Species differences in the conjugation of free bile acids with taurine and glycine. Biochem J 63: , Sjovall J: Dietary glycine and taurine on bile acid conjugation in man. Proc Soc Exp Bioi Med 100: , Encrantz J-C, Sjovall J: On the bile acids in duodenal contents of infants and children. Clin Chim Acta 4: , Dam H, Kruse I, Kallehauge HE, et al: Studies on human bile. I. Composition of bladder bile from cholelithiasis patients and surgical patients with normal bile compared with data for bladder bile of hamsters on different diets. Scand J Clin Lab Invest 18: , McLeod GM. Wiggins HS: Bile salts in small intestinal contents after ileal resection and in other malabsorption syndromes. Lancet 1: , Abaurre R, Gordon SG, Mann JG, et al: Fasting bile salt pool size and composition after ileal resection. Gastroenterology 57: , Bengmark S, Ekdahl P-H, Olsson R: Effect of taurine and glycine treatment on the conjugation of bile acids in partially hepatectomised rats. Acta Chir Scand 128: , Hofmann AF, Thomas PJ, Smith LH, et al: Pathogenesis of secondary hyperoxaluria in patients with ileal resection and diarrhea. (abstr). Gastroenterology 58:960, Dowling RH, Rose GA, Sutor DJ: Hyperoxaluria and renal calcili in ileal disease. Lancet 1: , Morris TQ: Diversity of biliary secretory mechanisms (abstr). J Clin Invest 49:68a, Erlinger S, Dhumeaux D, Benhamou J-P, et al: La secretion biliaire du lapin: preuves en faveur d'une important fraction independante des sels biliaires. Rev Fr Etud Clin Bioi 14: , Wheeler HO, Ramos OL: Determinants of the flow and composition of bile in the unanesthetised dog during constant infusions of sodium taurocholate. J Clin Invest 39: , Priesig R, Cooper HL, Wheeler HO: The relationship between taurocholate secretion rate