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p r f c t p p f c he regulation of genes that are essential to many hepatic metabolic and transport functions are medited in large part by the action of small molecules that unction as nuclear receptor ligands.1 This process has een labeled chemical genomics.2 These ligands bind to heir specific nuclear receptors, activating the receptor hat then binds to specific elements in a gene’s promoter esulting in stimulation or inhibition of gene expresion.3 In the liver many drugs, metabolites, and herbal ompounds exert their biologic properties as ligands for uclear receptors.4,5 Some familiar examples of drugs hat activate nuclear receptors include phenobarbital and t John’s wort. Both drugs induce hepatic drug metablizing enzymes by acting as ligands for the pregnane X eceptor (PXR), which binds to specific response eleents in the promoter of cytochrome P-450 3A CYP3A), a major hepatic microsomal drug–metabolizng enzyme. This induction then indirectly affects the etabolism and systemic effects of a wide variety of other ompounds metabolized by CYP3A. An extract of 4 ifferent plants, Yin Zhi Huang, has been used tradiionally for treating neonatal jaundice in China and is a igand for the constitutive androstane receptor (CAR) hat up-regulates the expression of several different liver ransporters and enzymes involved in the hepatic clearnce of serum bilirubin.5 Indeed, more than 10% of edically useful drugs are known to exert their biologic ehavior by binding to ligand-binding domains on speific nuclear receptors.4 Table 1 shows some of the most mportant nuclear receptors and their physiologic ligands hat determine the hepatic transport and metabolism of variety of xenobiotics and endogenous substrates. In this issue of GASTROENTEROLOGY, Marschall et al6 eport that rifampicin and ursodeoxycholic acid (UDCA), wo well-known drugs used in the treatment of cholesatic liver disease, each stimulate the transcription of a istinct set of genes that together decrease bile acid ptake, enhance bile acid detoxification and excretion, nd stimulate the clearance of bilirubin. Rifampicin inreased the expression of CYP3A4, uridine 5=-glucuonosyl transferase (UGT1A1), and multidrug-resisance–associated protein (MRP2) whereas UDCA timulated the bile salt export pump (BSEP), and mulidrug resistance protein (MDR3), and MRP4. Although hese effects were observed in patients undergoing choecystectomy who otherwise were healthy, these separate ut complementary effects on gene and protein expresion are predicted to have beneficial effects if they ocurred in patients with cholestatic liver disease. These ndings thus suggest that there is therapeutic benefit rom combining drugs such as rifampicin and UDCA hat target nuclear receptors that have coordinated effects n the transcriptional regulation of hepatobiliary excreory mechanisms.7 How does this occur? The hepatic clearance of bile acids and bilirubin can be ivided into 4 phases that include the following: phase 0, epatic uptake; phase I, metabolism (eg, hydroxylation); hase II, detoxification (eg, conjugation); and phase III, xcretion. Nuclear receptors and their ligands that are he major determinants of the functional expression of enes that determine these pathways are shown in Figure , together with enzymes that control bile acid synthesis. In the enterohepatic circulation, the hepatic uptake of onjugated bile acids (phase 0) is mediated predominantly y the sodium taurocholate cotransporting polypeptide TCP (SLC10A1), whereas unconjugated bile acid uptake s facilitated by several organic anion transporting polypepides (OATPs) on the sinusoidal membrane, but predomi

The cystic fibrosis transmembrane conductance regulator (CFTR) provides an important apical route for Cl(-) secretion across intestinal epithelia. A thiazolidinone-type CFTR blocker (CFTR(inh)-172) reduced cholera toxin-induced fluid accumulation in mouse intestinal loops. Here, we characterize the efficacy and pharmacodynamics of CFTR(inh)-172 in blocking cAMP and cGMP induced Cl(-)/fluid secretion in rodent and human intestine.CFTR(inh)-172 inhibited cAMP and cGMP agonist induced short-circuit current by95% in T84 colonic epithelial cells (K(I) approximately 3 micromol/L) and in mouse and human intestinal sheets (K(I) approximately 9 micromol/L). A single intraperitoneal injection of CFTR(inh)-172 (200 microg) blocked intestinal fluid secretion in a rat closed-loop model by90% for cholera toxin and70% for STa Escherichia coli toxin. In mice, CFTR(inh)-172 (20 microg) inhibited cholera toxin-induced intestinal fluid secretion by 90% (persistence t(1/2) approximately 10 hours, K(I) approximately 5 microg) and STa toxin by 75% (K(I) approximately 10 microg). Tissue distribution and pharmacokinetic studies indicated intestinal CFTR(inh)-172 accumulation facilitated by enterohepatic circulation. An oral CFTR(inh)-172 preparation reduced fluid secretion by90% in a mouse open-loop cholera model.A small molecule CFTR blocker markedly reduced intestinal ion and fluid secretion caused by cAMP/cGMP-dependent bacterial enterotoxins. CFTR inhibition may thus reduce fluid secretion in infectious secretory diarrheas.

Background & Aims: The mechanism for abnormal hepatic bile acid transport was investigated in an 18-month-old Amish boy who presented with pruritus, poor growth, and severe bleeding episodes. Serum bilirubin, γ-glutamyltranspeptidase, and cholesterol levels were normal, but prothrombin time and partial thromboplastin time were prolonged and bone alkaline phosphatase level was elevated. Methods and Results: Cholic acid plus chenodeoxycholic acid levels measured by capillary gas-chromatography were 32 times higher than control in serum (34.7 vs. 1.1 ± 0.4 μg/dL) but were not detected in liver and were reduced in gallbladder bile. Treatment with ursodiol, a more hydrophilic bile acid, improved pruritus, produced 37% weight gain, and after 2 years reduced serum primary bile acid concentrations about 85%, while accounting for 71% of serum and 24% of biliary bile acid conjugates. On ursodiol therapy, hepatic bile acid synthesis was enhanced 2-fold compared with controls, and microscopy revealed chronic hepatitis without cholestasis. Three younger sisters with elevated serum bile acids responded positively to ursodiol. Microsatellite markers for the FIC1 (gene for Byler's disease) region in these 4 children were inconsistent with linkage to FIC1 . Conclusions: Conjugated cholic acid and chenodeoxycholic acid were synthesized in the liver and secreted into bile but could not reenter the liver from portal blood and accumulated in serum. In contrast, unconjugated ursodiol entered the liver and was conjugated and secreted into bile. Thus, the enterohepatic circulation of all conjugated bile acids was interrupted at the hepatic sinusoidal basolateral membrane. Unconjugated ursodiol bypassed the hepatic uptake block to enlarge the biliary and intestinal bile acid pools. A mutation in FIC1 recognized among the Amish and linkage of the disorder to FIC1 were excluded. GASTROENTEROLOGY 2000;119:188-195

Background & Aims: Enterohepatic recirculation of nonsteroidal anti-inflammatory drugs is a critical factor in the pathogenesis of intestinal injury, but the underlying mechanism of toxicity remains obscure. The aim of this study was to examine the role of diclofenac acyl glucuronide, which is the major biliary metabolite and is chemically reactive, in the precipitation of small intestinal ulceration. Methods: Hepatocanalicular conjugate export pump-deficient (TR − ) rats were used to selectively block diclofenac enterohepatic circulation without interrupting bile flow. Bile from diclofenac-treated normal rats was orally transferred to wild-type and TR − rats, and the extent of ulcer formation was compared with that induced by control bile containing free diclofenac. The effect of induction of hepatic diclofenac glucuronosyltransferase on the severity of diclofenac-induced ulceration was also determined. Results: TR − rats were refractory to diclofenac given either intraperitoneally or perorally. However, transfer of bile containing diclofenac glucuronide significantly increased the extent of ulcer formation in both normal and TR − rats. Moreover, induction of glucuronosyltransferase aggravated intestinal ulceration. Conclusions: The reactive acyl glucuronide of diclofenac, or the acyl glucuronide of one of its oxidative metabolites, is directly involved in the pathogenesis of small intestinal injury. GASTROENTEROLOGY 1998;115:1476-1482

Background & Aims: Oral administration of ursodeoxycholic acid (UDCA) and cholesterol causes bile salt malabsorption; the former by competition for and the latter by down-regulation of ileal bile acid transporters. Because ileectomy in rats induces enterohepatic cycling of bilirubin, the hypothesis that dietary steroids might have the same effect was tested. Methods: Male inbred C57L/J mice and Sprague–Dawley rats were fed low doses of UDCA, chenodeoxycholic acid (CDCA), or cholesterol added to laboratory chow with simultaneous chow-fed controls. After 1 week (mice) or 2 weeks (rats), indices of bile salt malabsorption and enterohepatic cycling of bilirubin were measured, including bilirubin secretion rates into bile, serum and intestinal bilirubin and bile salt levels, and urobilinogen levels in cecum, large intestine, and feces. Results: Dietary UDCA and cholesterol, but not CDCA, significantly increased bilirubin secretion rates into bile. In UDCA-fed mice, gallbladder biles contained increased levels of bilirubin conjugates and unconjugated bilirubin, and in 60%, granules of amorphous calcium bilirubinate precipitated. Dietary cholesterol and bile acids, particularly UDCA, increased cecal bile salt levels, unconjugated bilirubin and urobilinogen concentrations, and decreased fecal bilirubin outputs, consistent with colonic absorption. Conclusions: By causing bile salt malabsorption, dietary UDCA and cholesterol induce enterohepatic cycling of bilirubin. GASTROENTEROLOGY 1998;115:722-732

BACKGROUND & AIMS: The pathogenesis of nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal damage remains poorly understood. The aim of this study was to examine the relative importance of the three suggested components of the pathogenesis of NSAID enteropathy, namely, epithelial permeability, enteric bacterial numbers, and enterohepatic recirculation, using an NSAID derivative (nitrofenac) that does not cause small intestinal damage. METHODS: Rats were given diclofenac or nitrofenac at 12-hour intervals. Epithelial permeability to [51Cr]-ethylenediaminetetraacetic acid and enteric bacterial numbers were determined after 1-4 doses of the drugs. Serum levels and biliary excretion of the two drugs were determined by high-performance liquid chromatography. RESULTS: Diclofenac caused a progressive increase in epithelial permeability, marked increases in enteric gram-negative bacterial numbers, and frank intestinal ulceration. Nitrofenac caused similar changes in intestinal permeability after a single dose but no further increase with repeated administration. Moreover, nitrofenac had no effect on enteric bacterial numbers and did not cause frank ulceration. Unlike diclofenac, nitrofenac did not undergo extensive enterohepatic recirculation. Two other NSAIDs that do not undergo enterohepatic recirculation (nabumetone and aspirin) also did not modify enteric bacterial numbers or cause intestinal ulceration. CONCLUSIONS: Enterohepatic recirculation of NSAIDs is of paramount importance in the pathogenesis of enteropathy caused by these drugs, whereas suppression of prostaglandin synthesis is relatively unimportant. (Gastroenterology 1997 Jan;112(1):109-17)

BACKGROUND & AIMS: Fasting increases serum bilirubin levels in both humans and rats. Because the pathogenesis of fasting hyperbilirubinemia is not fully understood, the effect of fasting on disposition of bile pigments was investigated in rats. METHODS: Bilirubin and urobilinogen were determined in excreta, bile, plasma, and liver tissues of fasted Gunn and Wistar rats. RESULTS: Fasting increased the intestinal transit time of Wistar rats. As a result, the fecal output of bile pigments was decreased by food deprivation. In contrast, the intestinal content of total bile pigments was augmented in both Wistar and Gunn rats. This finding was paralleled by the increase of serum bilirubin concentration in both rat strains. A similar increment of serum bilirubin levels was observed after injection of bilirubin into the cecum of Wistar rats. Furthermore, biliary efflux of bilirubin in Wistar rats was increased after 48 hours of fasting. Intubation of nonabsorbable bulk to fasted Wistar rats prevented the increase of serum bilirubin levels during a 48-hour period of food deprivation. CONCLUSIONS: Fasting decreases intestinal motility and elimination of bile pigments. Accumulation of bilirubin in the intestine during fasting allows enhanced enterohepatic circulation and results in an increased reflux to plasma. This seems to be a major factor involved in fasting-induced hyperbilirubinemia. (Gastroenterology 1996 Jul;111(1):217-23)

Background: Developmental changes in passive bile salt absorption may alter the enterohepatic circulation. Methods: 14-, 21-, and 40-day-old anesthetized male Sprague-Dawley rats were studied. Jejunum and ileum were isolated, cannulated, and injected or perfused with a taurocholate, [ 3 H]taurocholate, and nonabsorbable marker solution. Bile was collected. Results: Using bolus injection, jejunal taurocholate absorption rates and total taurocholate absorption were nonsaturable, linearly related to taurocholate dose, and decreased from 14 days (1.62 nmol · cm −1 · min −1 ) to 21 days (1.05 nmol · cm −1 · min −1 ) and 40 days (0.54 nmol · cm −1 · min −1 ). While total taurocholate absorption decreased (14 days, 52.4%; 21 days, 43.7%; 40 days, 30.5%), hepatic taurocholate clearance increased (14 days, 18.2%; 21 days, 23.7%; 40 days, 37.3%). Hepatic taurocholate clearance was saturated only at 14 days. Using jejunal perfusion, total taurocholate absorption (14 days, 62.0%; 21 days, 43.1%; 40 days, 45.3%) and taurocholate absorption rate decreased with age (14 days, 941.13 nmol · cm −2 · min −1 per micromole of taurocholate; 21 days, 411.28 nmol · cm −2 · min −1 per micromole of taurocholate; 40 days, 334.50 nmol · cm −2 · min −1 per micromole of taurocholate). Conclusions: Passive jejunal bile salt absorption and decreased hepatic bile salt clearance could account for the low intraluminal and high serum bile salt levels observed in immature animals and in human neonates.

The assimilation of labeled cobalamin and the transport of corrinoids in portal blood, peripheral venous blood, and bile were studied in eight cholecystectomized patients, after ingestion of a dose of cyano[57Co]cobalamin (0.5 microCi). The radioactivity appeared in the portal vein after a delay of 1.5-2 hours and in the peripheral vein 1 hour later. In bile, it reached a maximum at 24-72 hours; the excreted cobalamin corresponded to 1.42% +/- 0.92% of the dose ingested. The output of total corrinoids was 1.85 nmol/day. The high-performance liquid chromatography analysis of bile showed the presence of methylcobalamin, 5'-deoxyadenosylcobalamin, hydroxocobalamin, and an unknown corrinoid. This corrinoid bound to R binder but not to the intrinsic factor, and it had the same retention time as cobinamide. The R binder was the single cobalamin-binding protein found in bile. It was completely saturated in some periods of bile secretion. The corrinoids corresponding to such a period were eluted in Sephacryl S 300 gel filtration (Pharmacia Fine Chemicals, Uppsala, Sweden) in two peaks corresponding to saturated R binder and to free cobalamin. The mean level of total corrinoid was significantly higher in the portal vein (593 +/- 238 pmol/L) than in the peripheral vein (376 +/- 114 pmol/L) (P less than 0.01). This "cobalamin analogue" fraction was hypothetical because it was calculated from the difference between total corrinoid concentration and the so called "true cobalamin" concentration. This difference corresponded to the cobalamin analogue fraction. These data show that bile removes not only cobalamin but also cobalamin analogues and that R binder is the single carrier protein involved in their excretion.

To test the effect in rodents of chronic ingestion of a bile acid resistant to deconjugation, cholylsarcosine was synthesized and its transport, metabolism, and effect on biliary bile acid and biliary lipid composition were determined in rabbits, hamsters, and rats. Cholylsarcosine was shown to be well absorbed from the ileum but underwent little absorption from the jejunum or colon. When cholylsarcosine was administered in the diet at 140 μmol/kg · day, it was well absorbed and underwent little biotransformation during enterohepatic cycling; however, both bacterial deconjugation and dehydroxylation (without deconjugation) occurred to a small extent. With chronic feeding, cholylsarcosine accumulated to compose 24%–29% of circulating bile acids in all 3 rodent species. It was rapidly lost from the enterohepatic circulation, with a daily fractional turnover rate of 75%–150%, depending on the species. Cholylsarcosine caused no change in liver tests or hepatic morphology and did not influence biliary lipid secretion. When cholyltaurine was fed, it was also absorbed, but, in contrast to cholylsarcosine, was rapidly deconjugated and dehydroxylated to form deoxycholic acid. The deoxycholic acid accumulated in the enterohepatic circulation, as evidenced by a slow fractional turnover rate of 26%–40% per day, depending on the species. It is concluded that cholylsarcosine is absorbed from the ileum, has an enterohepatic circulation, does not undergo appreciable deconjugation or dehydroxylation in these rodents, and is nontoxic. In the rodent, the circulating bile acids can be somewhat enriched when a bile acid resistant to deconjugation is ingested; but the effect on the steady state biliary bile acid composition is less than that obtained when cholyltaurine is administered because cholyltaurine is biotransformed to deoxycholic acid, which in turn is absorbed and has its own efficient enterohepatic circulation.

Background: Total parenteral nutrition (TPN) is a life-saving therapy in critically ill patients. Unfortunately, it is associated with significant morbidity and mortality, including gut atrophy and Parenteral Nutrition Associated Liver Disease. A lack of enteral feeding during TPN, disrupts key signaling molecules and the enterohepatic circulation, which appears to be a major contributor to disease pathology. Our group previously demonstrated thatenterally administeredbile acid,oleanolic acid (OA)significantly improved gut density andmorphology. OAis a ligand for the G-protein coupled receptor TGR-5. TGR-5 is known to induce transcription of glucagon-like peptide (GLP)-1 and GLP-2. While GLP-1 regulates hepatic steatosis and inflammation, GLP2 is involved in gut growth and motility.Hypothesis: We hypothesize that TPN infusion disrupts the TGR5-GLP axis and exogenous GLP-1, GLP-2 infusion will ameliorate TPN induced injury. Methods: Seven to ten day old piglets were implanted with jugular venous (JVC) and duodenal catheters (DC). Animals were randomized to receive Enteral Feeding (EN) or TPN. The TPN group was subdivided into TPN plus GLP-1 and GLP-2 (n=3). Milk was delivered via the DC and TPN was infused via the JVC. GLP-1 and GLP-2 were dosed at3 mcg/kg/day and 30 mcg/kg/day respectively. Results: Mean daily weight gain (grams) for control Enteral Fed (EN) vs TPN animals and standard deviation (±SD) was 102.4±10.8, 91.03±12.1 respectively, p>0.05. Marked gut atrophy was noted with TPN.Mean gut density as measured in grams/cm of gut tissue and standard deviation (±SD) in EN vs TPN was 0.35±0.03, 0.185±0.05, p

Background: Total parenteral nutrition (TPN) is a life-saving therapy in critically ill patients. Unfortunately, it is associated with significant morbidity and mortality, including gut atrophy and Parenteral Nutrition Associated Liver Disease. A lack of enteral feeding during TPN, disrupts key signaling molecules and the enterohepatic circulation, which appears to be a major contributor to disease pathology. Our group previously demonstrated thatenterally administeredbile acid,oleanolic acid (OA)significantly improved gut density andmorphology. OAis a ligand for the G-protein coupled receptor TGR-5. TGR-5 is known to induce transcription of glucagon-like peptide (GLP)-1 and GLP-2. While GLP-1 regulates hepatic steatosis and inflammation, GLP2 is involved in gut growth and motility.Hypothesis: We hypothesize that TPN infusion disrupts the TGR5-GLP axis and exogenous GLP-1, GLP-2 infusion will ameliorate TPN induced injury. Methods: Seven to ten day old piglets were implanted with jugular venous (JVC) and duodenal catheters (DC). Animals were randomized to receive Enteral Feeding (EN) or TPN. The TPN group was subdivided into TPN plus GLP-1 and GLP-2 (n=3). Milk was delivered via the DC and TPN was infused via the JVC. GLP-1 and GLP-2 were dosed at3 mcg/kg/day and 30 mcg/kg/day respectively. Results: Mean daily weight gain (grams) for control Enteral Fed (EN) vs TPN animals and standard deviation (±SD) was 102.4±10.8, 91.03±12.1 respectively, p>0.05. Marked gut atrophy was noted with TPN.Mean gut density as measured in grams/cm of gut tissue and standard deviation (±SD) in EN vs TPN was 0.35±0.03, 0.185±0.05, p

Background & Aims: Release of neurotensin (NT) from intestines is markedly stimulated by ingested fat, and NT may facilitate lipid digestion and absorption through various actions that are not fully understood. Our recent finding that NT stimulates hepatic output of bile acids only when bile delivery to the intestine is maintained has led us to investigate the effects of NT on bile acid absorption in the rat small intestine. Methods: We measured the effects of intravenous infusion of NT (3-10 pmol · kg−1 · min−1) on biliary recovery of 3H-taurocholate (3H-TC) and 3H-cholate administered into proximal and distal intestines or into isolated intestinal segments in situ in biliary fistula rats. To further understand the underlying mechanisms involved, the effects of NT on intestinal absorption of 3H-D-glucose, 3H-leucine, 14C-antipyrine, and 51Cr-EDTA were investigated by monitoring the absorption of radioactivity into superior mesenteric venous blood. Results: Infusion of NT, at doses that caused near physiologic increases in blood NT levels, increased biliary recovery of 3H-TC from the jejunum (3.4-fold) and ileum (1.7-fold), but did not enhance absorption of 3H-cholate. NT also facilitated transcellular uptake of 3H-glucose and 3H-leucine and increased paracellular uptake to 51Cr-EDTA and 3H-mannitol, but did not alter the absorption rate for 14C-antipyrine. Conclusions: These results indicate that NT can exert a facilitative effect on intestinal bile acid absorption and return to liver. This effect of NT may involve increases in paracellular absorption and carrier-mediated transport by mechanisms not yet identified. GASTROENTEROLOGY 2001;120:151-160

Background & Aims: Enteropathy is a frequent complication of diclofenac and other nonsteroidal anti-inflammatory drugs, yet little is known about the underlying mechanism. One possibility is that reactive metabolites of diclofenac form adducts with enterocyte macromolecules, as previously shown for liver. We addressed this possibility by using immunohistochemistry to detect diclofenac adducts. Methods: Rats were treated orally with diclofenac (10–100 mg/kg) and killed after 1–24 hours, and their gastrointestinal (GI) tracts were evaluated for ulcer number and area. Adduct distribution and intensity were assessed by immunohistochemistry by using a technique to simultaneously process and stain multiple intestinal rings. Results: Drug treatment led to dose-dependent formation of both adducts and ulcers only in small intestine and only in animals with intact enterohepatic circulation. Adducts formed within enterocytes by 1 hour, translocated to the brush border, preceded ulceration and vascular protein leakage, and were intense at sites of ulceration. Adducts and ulcers exhibited a parallel distribution within intestinal quintiles: 3rd > 5th >> 1st. Conclusions: Diclofenac treatment resulted in the formation of drug adducts in enterocytes. Because this molecular change occurred before ulceration, was dose dependent, and exhibited concordant distribution with extent of ulceration, the results suggest a causal role for drug adduct formation in diclofenac enteropathy. GASTROENTEROLOGY 2000;119:1537-1547

Background & Aims: Patients with ileal disease, bypass, or resection are at increased risk for developing gallstones. In ileectomized rats, bilirubin secretion rates into bile are elevated, most likely caused by increased colonic bile salt levels, which solubilize unconjugated bilirubin, prevent calcium complexing, and promote its absorption and enterohepatic cycling. The hypothesis that ileal disease or resection engenders the same pathophysiology in humans was tested. Methods: Sterile gallbladder bile samples were obtained intraoperatively from 29 patients with Crohn's disease and 19 patients with ulcerative colitis. Bilirubin, total calcium, biliary lipids, β-glucuronidase activities, and cholesterol saturation indices in bile were measured, and markers of hemolysis and ineffective erythropoiesis in blood were assessed. Results: Bilirubin conjugates, unconjugated bilirubin, and total calcium levels were increased 3-10-fold in bile of patients with ileal disease and/or resection compared with patients with Crohn's colitis or ulcerative colitis. Biliary bilirubin concentrations correlated positively with the anatomic length and duration of ileal disease. Endogenous biliary β-glucuronidase activities were comparable in all groups, and both the hemogram and serum vitamin B12 levels were normal. Conclusions: This study establishes that increased bilirubin levels in bile of patients with Crohn's disease are caused by lack of functional ileum, supporting the hypothesis that enterohepatic cycling of bilirubin occurs. GASTROENTEROLOGY 1999;116:1420-1427

BACKGROUND & AIMS: Occurrence of hyperbilirubinemia after fasting has been recognized for many years. The pathogenesis of this syndrome is unclear. Although recent studies suggest that increased intestinal deconjugation and reabsorption of bilirubin may play a major role in establishment of hyperbilirubinemia during fasting, other studies have suggested that fasting down-regulates intrinsic hepatocyte transport of bilirubin. The present study was designed to examine this possibility in the isolated perfused rat liver. METHODS: Transport of 3H-bilirubin and 35S-sulfobromophthalein (BSP) was examined in isolated perfused livers from 48-hour fasted or control rats using a multiple indicator dilution technique. Data were analyzed to quantify single-pass extraction (model-independent analysis) and were also fit by computer to the model of Goresky to quantify unidirectional fluxes. RESULTS: Fasting for 48 hours resulted in an approximately 40% reduction in liver weight but had no effect on model-dependent or model-independent parameters of transport. Despite the fact that the liver was smaller, single-pass extraction of bilirubin and BSP by livers from fasted animals did not differ from control, indicating a greater efficiency at uptake of bilirubin and BSP. CONCLUSIONS: Enhanced enterohepatic circulation of bilirubin, not altered hepatic transport, is a major factor in the pathogenesis of fasting-induced hyperbilirubinemia. (Gastroenterology 1997 Nov;113(5):1707-13)

Bile Acid Malabsorption (BAM) resulting from bile acid overproduction or reduced transport can increase fecal bile acids and cause chronic diarrhea. BAM is thought to account for up to 30% of patients diagnosed with irritable bowel syndrome (IBS-D). Current methods of diagnosing BAM are limited; it is likely under-diagnosed. Our objective was to develop a non-invasive method of detecting BAM by synthesizing and testing a multi-fluorinated bile acid (MFBA) that can be used for fluorine magnetic resonance imaging (MRI) of bile acid enterohepatic circulation. A prototype MFBA was synthesized by conjugating trifluoroacetyl lysine to cholic acid (19F-CA-lys). This MFBA was tested for transport and inhibition of the apical sodium dependent bile acid transporter (ASBT) using stably transfected MDCK cell monolayers and of Na+/taurocholate cotransporting polypeptide (NTCP) using stably transfected HEK cell monolayers. In Vitro, 19F-CA-lys was a potent inhibitor and substrate of both ASBT (Ki = 20.0±3.9 μM, Kt = 39.4±23.8 μM, normalized Vmax =0.853 ± 0.197) and NTCP (Ki = 2.93±0.60 μM, Kt = 8.99 ± 2.79 μM, normalized Vmax = 0.281±0.052). Stability of 19F-CA-lys (5 μM) was measured after treatment with Caco-2 cell homogenates, rat plasma, rat liver microsomal S9 pools, simulated intestinal fluid with pancreatic enzymes, 1 M HCl, buffers at pH 7.4 and 6.8, and choloylglycine hydrolase (CGH) (N=3 for each condition). Except for CGH, 19F-CA-lys was stable in each condition. After 1 h in CGH, 60.4±4.0% 19F-CA-lys remained compared to 2.4±0.5% naturally-occurring taurocholic acid (P

BACKGROUND/AIM: Evaluation of bile acids (BA) is a useful method for assessing changes of the intestinal flora in patients with ulcerative colitis (UC). During enterohepatic circulation, conjugated BA is deconjugated to free BA by intestinal bacteria. The presence of intestinal microflora (Clostridium and Eubacterium) leads to 7 alpha-dehydroxylation of cholic acid (CA) and chenodeoxycholic acid (CDCA), yielding deoxycholic acid (DCA) and lithocholic acid, respectively. It was reported that the Lachnospiraceae subgroup of Firmicutes (including Clostridium) are decreased in the colon of UC patients compared to controls without inflammatory bowel disease. We have already reported that the serum%CDCA is significantly higher in patients with UC than in healthy volunteers (HV), while serum%DCA is significantly lower in UC patients than in HV, and these changes do not depend on the activity or extent of UC. The aim of the present study was to elucidate the effects of probiotics in patients with UC by examining the serum BA profile. PATIENTS/METHODS: The study population was 27 patients in whom UC was diagnosed from endoscopic and histological findings. All patients underwent ileocolonoscopy with appropriate biopsies. They were divided into the following 2 groups based on endoscopic findings: 15 patients with distal UC (dUC) and diffuse changes extending from the rectum to the splenic flexure, and 12 patients with more extensive UC or pancolitis (pUC). Treatment was givenwithmesalazine or salazosulfapyridine (5-ASA) and all patients achieved remission. After entering remission, they were treated with by 5-ASA plus the probiotic Clostridium butyricum Miyairi (MIYA) (3 g/day) for 4 weeks. The control group was composed of 8 HV. Routine laboratory tests were performed on the basis of clinical need, while fasting serum samples for measurement of BA were obtained before and after treatment with 5-ASA plus MIYA for 4 weeks. Serum BA fractions were analyzed by HPLC. RESULTS: There were no significant differences of serum total BA among the 3 groups. Before treatment, %CDCA was significantly higher in the dUC and pUC groups than in the HV group, while %DCA was significantly lower than in the HV group. In the pUC group, %CDCA was significantly higher and %DCA was significantly lower than in the HV group after 4 weeks of probiotic treatment. In the dUC group, however, there was no significant difference of %CDCA or %DCA after 4 weeks compared with the HV group. There were no significant differences in the ratio of conjugated BA to total serum BA among the three groups. CONCLUSIONS: These results suggest that intestinal bacteria involved in the deconjugation of BA are restored when patients achieve remission of UC by treatment with 5-ASA, and thatMIYA restoredmicroflora involved in 7 alpha-dehydroxylation in the dUC group, but not in the pUC group.

Background: Cholylsarcosine, the synthetic conjugate of cholic acid and sarcosine, is resistant to deconjugation-dehydroxylation during enterohepatic cycling in rodents and improves lipid absorption in a canine model of intestinal bile acid deficiency caused by distal intestinal resection. Experiments were performed to define its metabolism and effect on biliary secretion in humans. Methods: The circulating bile acid pool was labeled with [ 14 C]cholylsarcosine, and its turnover rate and biotransformation were determined by sampling bile daily. Cholylsarcosine (or cholyltaurine) was infused into the duodenum for 8 hours to define its effect on bile flow and biliary lipid secretion. Results: Cholylsarcosine was lost rapidly from the enterohepatic circulation with a t12 of 0.5 days. The compound was not biotransformed by hepatic or bacterial enzymes. Cholylsarcosine had choleretic activity similar to that of cholyltaurine but induced more phospholipid and cholesterol secretion than cholyltaurine in four of five subjects. Infusion of cholylsarcosine (or cholyltaurine) at a rate averaging 0.6 μmol · min −1 · kg −1 gave a biliary recovery of 0.2 μmol · min −1 · kg −1 ; this value is the T max for active ileal transport of conjugated bile acids in humans. Laboratory tests for liver injury remained within normal limits. Conclusions: In humans, cholylsarcosine is not metabolized, is nontoxic, and has similar effects on biliary secretion as cholyltaurine. It appears safe to test in long-term studies the effect of cholylsarcosine on bile acid-deficiency states in humans.

Background: A3309 modulates the enterohepatic circulation of bile acids (BA) by inhibiting the intestinal bile acid transporter (IBAT). This will result in an increased concentration of BA in the colon. The synthesis of bile acids in the liver can be measured indirectly by plasma concentrations of the intermediate 7-α-hydroxy-4-cholesten-3-one (C4). Methods: In Vitro part: A3309 potency was tested in transfected HEK293 cells. The cells expressing the various BA transporters were grown in Cytostar 96 w Scintillation Proximity Assay plates and the effect of increasing concentrations of A3309 on accumulation of 30 μM of radiolabelled glycocholic acid was monitored. In Vivo part: In dogs, constipation was induced by shifting the standard diet to ground beef dosed once daily until the faeces weight was

Hepatocyte dysfunction eventually results in the loss of canalicular bile formation. Without canalicular flow, intestinal bile acid may originate from plasma by reverse transport. Anhepatic rats with preserved intestinal function permit evaluation of such transport. In the present study, plasma taurocholate clearance was markedly decreased in anhepatic rats. The relative proportion of free cholate increased with time. Peripheral tissues contained virtually only cleared taurocholate, but the intestinal contents were mainly free cholate. This indicates the intestinal contents as the source of the plasma cholate and shows an equilibrium between intestinal and plasma bile acid even without bile flow. The enteral administration of an anion exchange resin to anhepatic rats increased intestinal bile acid recovery and decreased the bile acid recovery in tissue. Plasma bile acid concentration was decreased and fractional loss increased threefold, confirming the anhepatic plasma-intestine bile acid equilibrium. However, the enhanced plasma clearance produced by the resin was less than 1% of the fractional loss found in the intact rat. These data show a very limited bile acid flux between intestine and plasma without bile flow, which could be modestly influenced by an intestinal bile acid sequestrant.

The role of enterohepatic circulation and specific bile acids in the initiation and caudad migration of duodenal migrating motor complexes (MMCs) was investigated in conscious dogs. All dogs had spontaneous duodenal MMCs that migrated to the terminal ileum when bile flow was intact. During the first 3 days after total external biliary diversion, no MMCs originated in the duodenum. Instead, all MMCs originated in the jejunum and migrated to the ileum. During the next 4 days of total external biliary diversion, 81% of the MMCs originated in the jejunum and 19% in the duodenum. When normal bile flow was restored after 9 days of total external biliary diversion, regular duodenal MMCs resumed after a delay of 126 ± 27 minutes. Perfusion of individual bile acids or dogs' own bile, but not saline or alkaline solution, into the duodenum or perfusion of dogs' own bile directly into the ileum during total external biliary diversion restarted duodenal MMCs with a time lag of about 2 hours. The authors conclude that intact enterohepatic circulation is essential for the initiation of regular duodenal MMCs.

The purpose of this study was to develop and validate a method of directly measuring ileal bile acid absorption efficiency during a single enterohepatic cycle (first-pass ileal clearance). This has become feasible for the first time because of the availability of the synthetic gamma-labeled bile acid 75Selena-homocholic acid-taurine (75SeHCAT). Together with the corresponding natural bile acid cholic acid-taurine (labeled with 14C), SeHCAT was infused distal to an occluding balloon situated beyond the ampulla of Vater in six healthy subjects. Completion of a single enterohepatic cycle was assessed by obtaining a plateau for 75SeHCAT activity proximal to the occluding balloon, which prevented further cycles. Unabsorbed 75SeHCAT was collected after total gut washout, which was administered distal to the occluding balloon. 75SeHCAT activity in the rectal effluent measured by gamma counter was compared with that of absorbed 75SeHCAT level measured by gamma camera and was used to calculate first-pass ileal clearance. This was very efficient (mean value, 96%) and showed very little variation in the six subjects studied (range, 95%-97%). A parallel time-activity course in hepatic bile for 14C and 75Se during a single enterohepatic cycle, together with a ratio of unity for 14C/75Se in samples obtained at different time intervals, suggests that 75SeHCAT is handled by the ileum like the natural bile acid cholic acid-taurine. Extrapolation of 75SeHCAT first-pass ileal clearance to that of the natural bile acid therefore seems justifiable. In a subsidiary experiment, ileal absorption efficiency per day for 75SeHCAT was also measured by scanning the gallbladder area on 5 successive days after the measurement of first-pass ileal clearance. In contrast with absorption efficiency per cycle, absorption efficiency per day varied widely (49%-86%), implying a possible wide variation in recycling frequency per day.

The scavenger receptor class B type I (SR-BI), which is expressed in the liver and intestine, plays a critical role in cholesterol metabolism in rodents. While hepatic SR-BI expression controls high density lipoprotein (HDL) choles- terol metabolism, intestinal SR-BI has been proposed to facilitate cholesterol absorption. To evaluate further the rel- evance of SR-BI in the enterohepatic circulation of choles- terol and bile salts, we studied biliary lipid secretion, he- patic sterol content and synthesis, bile acid metabolism, fecal neutral sterol excretion, and intestinal cholesterol ab- sorption in SR-BI knockout mice. SR-BI deficiency selec- tively impaired biliary cholesterol secretion, without con- comitant changes in either biliary bile acid or phospholipid secretion. Hepatic total and unesterified cholesterol con- tents were slightly increased in SR-BI-deficient mice, while sterol synthesis was not significantly changed. Bile acid pool size and composition, as well as fecal bile acid excretion, were not altered in SR-BI knockout mice. Intestinal choles- terol absorption was somewhat increased and fecal sterol excretion was slightly decreased in SR-BI knockout mice rel- ative to controls. These findings establish the critical role of hepatic SR-BI expression in selectively controlling the utilization of HDL cholesterol for biliary secretion. In con- trast, SR-BI expression is not essential for intestinal choles

Studies were carried out in healthy subjects to characterize the enterohepatic circulation of lithocholate and its metabolites. When mixed with bile and infused into the jejunum, radiolabeled lithocholylglycine was absorbed more rapidly and more efficiently than sulfolithocholylglycine, based on recovery from bile. When these metabolites were administered at 1800 hr in a liquid test meal containing radiolabeled taurocholate as an absorbable marker, 60% of lithocholylglycine was conserved, based on recovery of radioactivity in fasting bile the following morning, but only 20% of sulfolithocholylglycine was conserved. Iotope dilution studies in 4 subjects showed that daily input of lithocholate into the bile acid pool averaged 100 mg per day, about one-third to one-half of the chenodeoxycholic acid synthesis, but the t 1/2 was extremely short (0.74 day). The small lithocholate pool (about 100 mg) could be explained by rapid fecal excretion caused by sulfation which decreases passive absorption in the jejunum and active absorption in the ileum. Experiments with [35S]sulfo- [3H]lithocholylglycine indicated little desulfation during enterohepatic cycling but rapid desulfation in the distal intestine, with absorption of 35S (presumably as sulfate) followed by urinary excretion. A decreasing 35S:3H ratio in bile indicated that some steroid moiety was conserved to be resulfated. These studies indicate that considerable lithocholate is absorbed from the distal intestine in healthy subjects but efficient sulfation results in rapid fecal excretion, so that the total lithocholate pool remains small. A multicompartment model, previously used to describe the metabolism of the steroid and amino acid moieties of the major conjugated biliary bile acids, was extended to encompass lithocholyl conjugates and their sulfates.

We studied the degree of interruption of the enterohepatic circulation of bile salts in patients with bile salt malabsorption after ileal resection, using a double isotope method to circumvent the problems due to absence of steady state conditions. We measured the proportion of the isotopically labeled pool of glycocholate and glycochenodeoxycholate present before a standard evening meal, which was still present in fasting bile the following morning. The proportion of glycocholate returned by the patients during this period averaged 5.8% of that returned by the control subjects, while the equivalent value for glycochenodeoxycholate was 2.3%. We conclude that in patients with ileal resection, the remaining small bowel is unable to contribute significantly in maintaining enterohepatic circulation of conjugated bile salts. In these patients, the bulk of the bile salts in the gallbladder before breakfast had been synthesized overnight.

A study was carried out to define the effect of somatostatin on bile secretion using a chronic bile fistula dog preparation. Bile flow, erythritol clearance, and biliary electrolyte and lipid composition were measured under steady-state conditions during four randomized treatments: placebo, somatostatin, cimetidine, and cimetidine + somatostatin. In addition, the effect of three increasing doses of secretin was defined during each treatment. A constant cholyltaurine infusion was used to compensate for the interrupted enterohepatic circulation of bile acids. Somatostatin caused a consistent reduction in total bile flow and biliary bicarbonate output, but its effect on canalicular bile flow, as assessed by erythritol clearance, was variable. Outputs of bile acids, lecithin, and cholesterol remained unchanged during the somatostatin infusion. Because the bile/plasma ratio of erythritol concentration increased greatly during somatostatin infusion, the reduction in bile flow was attributed to enhanced ductular reabsorption (or alternatively, decreased ductular secretion) of a fluid whose calculated electrolyte composition was identical to that induced by secretin infusion in the control group. Secretin and somatostatin antagonized each other's effects: at a low dose of secretin, somatostatin caused decreased bile volume, whereas the highest dose of secretin effectively antagonized the effect of somatostatin. The anticholeretic effect of somatostatin was not due to the inhibition of endogenous secretin, since cimetidine treatment did not influence the effect of somatostatin on bile flow or bicarbonate secretion. Further, the anticholeretic effect of somatostatin was not vagally mediated, since the infusion of an anticholinergic agent did not alter the effects of somatostatin. It was concluded that, in the dog, the major effect of a pharmacologic dose of somatostatin on bile flow is an anticholeresis caused by either an enhanced ductular reabsorption or inhibition of a ductular secretion of a bicarbonate-rich electrolyte fraction of bile, and that this effect is likely to be mediated by a local effect of somatostatin on the biliary ductule. Thus, in the dog, somatostatin and secretin both evoke ductular modification of bile; however, the effects of the two hormones are exactly opposite to each other.

A primate model employing awake cholecystopexed baboons has been designed to permit quantitative studies of gallbladder function and its influence on the hepatic secretion of bile salts. Duodenal perfusion with [3H]inulin measures total bile salt output into the duodenum, while periodic percutaneous sampling of gallbladder bile labeled with [14C]inulin permits quantitation of changes in volume and bile composition in the gallbladder. The appearance of [14C]inulin in the duodenum quantitates gallbladder emptying. The presence of the catheter does not change the gallbladder’s ability to concentrate either radiographic dye or bile salts, nor does it interfere with or induce contraction. During fasting, hepatic bile salt secretion was low (about 5 mmoles per min) with less than one-half the output entering the gallbladder. No gallbladder contents appeared in the duodenum until contraction was induced by cholecystokinin. However, after gallbladder contraction with mobilization of the bile salt pool, hepatic bile salt secretion increased 2- to 4-fold. The preliminary data suggest that gallbladder filling and emptying play a role in governing the movement of bile salts around the enterohepatic circulation.

Two boys with congenital diarrhea, steatorrhea, and growth failure were studied. Preliminary investigations indicated that the enterohepatic circulation of bile acids was interrupted. Radiographically, ileal structure was normal; ileal function was normal when assessed by vitamin B 12 absorption. To confirm our clinical suspicion that the patients had an isolated defect of ileal active bile acid transport, peroral terminal ileal biopsies were performed. Ileal mucosa was incubated in vitro in oxygenated Krebs-Ringer bicarbonate buffer containing 10 mM glucose and 0.1, 1.0, or 10.0 mM taurocholic acid at 37°C. Ileal uptake from the patients was 0.10 and 0.34 μmol/g dry wt · min in 0.1 mM taurocholic acid, 1.20 and 2.39 μmol/g dry wt · min in 1.0 mM taurocholic acid, and 21.19 and 11.14 μmol/g dry wt · min in 10.0 mM taurocholic acid. At every concentration, significant (p x ¯ ± SEM) at 0.1 mM; 6.36 ± 1.33 μmol/g dry wt · min at 1.0 mM, and 76.20 ± 19.30 μmol/g dry wt · min at 10.0 mM taurocholic acid. Ultra-structural examination of the ileal mucosa failed to demonstrate a significant structural abnormality. Significant reduction in ileal uptake of taurocholic acid accompanying clinical and biochemical findings of interruption of the enterohepatic circulation in the absence of mucosal disease suggests that these children have a previously undescribed, congenital transport defect that includes absence of active ileal bile acid transport presenting as diarrhea in infancy.

Radioactive lithocholate was administered intravenously to female rhesus monkeys with an exteriorized enterohepatic circulation, and the chemical form of biliary radioactivity was defined by thin layer chromatography. Most radioactivity was excreted in bile in the form of unsulfated conjugates (77%), indicating impaired sulfation relative to published data for man. Defective sulfation of lithocholate, the major bacterial biotransformation product of chenodeoxycholic acid, can explain accumulation of lithocholate in the enterohepatic circulation and provide a possible mechanism for the hepatotoxicity observed during chenodeoxycholic acid administration to the nonhuman primate.

A multicompartment model is described for the enterohepatic circulation of conjugated bile acids in man which encompasses bile acid synthesis, amino acid conjugation, bacterial deconjugation, hepatic reconjugation, and bacterial dehydroxylation. When the model was used to predict the fate of administered ring-labeled bile acid, there was good agreement between observed and calculated values. The model was also used to assess the validity of the customary single-compartment analysis of bile acid specific activity decay curves. The errors in estimates of pool size and synthesis rate were found to be small if the Lindstedt procedure was used (tracer administered as free bile acid and specific activity measurements made on combined conjugates of that acid). However, if tracer was given as a conjugated bile acid and the specific activity was determined on only that conjugate, the errors potentially were unacceptably large, especially if cholyltaurine was used. Several novel functions were defined for the enterohepatic circulation of conjugated bile acids, and these were used to derive equations describing biliary bile acid composition for both steroid and amino acid moiety in terms of hepatic and intestinal factors. The model should be of value for quantitative characterization of bile acid metabolism in health and disease.

Biliary excretion of cobalamin was studied in baboons after intravenous injection of 57 Co-radio-labeled cyanocobalamin. Radioactivity appeared in bile after 20 min, and up to 4.4% of the dose was recovered in 6 h. It was estimated that 4 μg cobalamin traverses the baboon biliary tract each day. These studies in the nonhuman primate confirm that there is considerable biliary excretion of cobalamin and that enterohepatic circulation of the vitamin is important for maintenance of normal cobalamin balance.

As food in the intestine "drives" the enterohepatic circulation and bile acids influence bile flow, we postulated that the cholestasis of total parenteral nutrition might be due to bile acid changes, and the cholelithiasis and biliary sludge of total parenteral nutrition to bile lipid changes. We therefore studied bile acid and bile lipid metabolism in the following groups of rats, with and without bile fistula: (a) nonfasted, orally fed controls, (b) orally fed controls fasted for 20 h, and (c) after 7 days of total parenteral nutrition. Biliary bile acid concentration (35.4 +/- 2.5 mM) and secretion (253 +/- 20.0 mumol/100 g body wt.24 h) increased significantly in the rats on TPN and the rats fasted for 20 h (38.8 +/- 2.5 and 243 +/- 23.4 mM, respectively) when compared with the orally fed controls (26.5 +/- 2.5 and 178 +/- 23.5 mM, respectively). Bile flow, however, was unchanged. Bile acid pool size (Eriksson washout technique) also increased from 43.4 +/- 3.0 mumol/100 g body wt in the controls to 50.5 +/- 4.8 in the group fasted for 20 h and 65.6 +/- 5.3 in the TPN group (p less than 0.05-0.01). Similar bile acid pool sizes (carcass extraction method) were found in the nonfistulated animals. Biliary cholesterol secretion and saturation were significantly less in the TPN rats than in the other two groups. Liver microscopy indicated only minimal fatty change, but serum bile acid and alkaline phosphatase levels were increased in the TPN group (p less than 0.05). Thus, during TPN bile acids stagnate within the enterohepatic circulation, increasing biliary bile acid concentration and secretion rates and expanding the pool size. However, the absence of an associated choleresis, together with abnormal liver function tests, suggest that alterations in bile acid metabolism cause a relative cholestasis in this model.

The metabolism of intravenously injected radiolabeled lithocholate, lithocholylglycine, and their 3a-sulfate esters was characterized in healthy subjects. Lithocholate radioactivity was excreted rapidly and predominantly in bile; the excreted radioactivity had the chromatographic properties of glycine and taurine conjugates of lithocholate, of which 60% were sulfated. Lithocholylglycine also was excreted rapidly and predominantly in bile, and 60% of excreted radioactivity was sulfated. Sulfolithocholate radioactivity was only partially conjugated (about 60%) in association with biliary excretion. Suifolithocholylglycine was excreted unchanged in bile. Neither sulfated derivative showed appreciable excretion in urine, although both were excreted more slowly in bile than unsulfated free or conjugated lithocholate. The data suggest that unconjugated lithocholate which is absorbed is completely conjugated and partially sulfated before excretion which occurs exclusively in bile. Since sulfation is not complete, some unsulfated lithocholate is always present in bile. This conjugated but unsulfated lithocholate, if reabsorbed, would be again partially sulfated during its next enterohepatic circulation. Thus, the end result of these biotransformations would be for absorbed lithocholate to be excreted in bile mostly, but not entirely as the sulfated conjugates.

The "apparent" fractional turnover rate of the gamma-labeled bile acid analogue 75selenohomocholic acid-taurine (75SeHCAT) was assessed from decline in radioactivity over the gallbladder area on 4 successive days using a gamma-camera, and was compared in the same subjects with the fractional turnover rate of the corresponding natural bile acid, cholic acid-taurine, labeled with 14C ([14C]CAT) using the classical Lindstedt technique. Very similar results were obtained in 5 healthy individuals (coefficient of variation 4.8%, medians 0.35 and 0.34, respectively). By contrast, the fractional deconjugation rate assessed from zonal scanning of glycine- and taurine-conjugated bile acids on thin-layer chromatography was much less for 75SeHCAT than for [14C]CAT (0.02 and 0.13, respectively; p less than 0.05). The fractional rate for deconjugation plus dehydroxylation was also determined by zonal scanning, and gave lower values for 75SeHCAT than for [14C]CAT (0.02 and 0.12, respectively; p less than 0.05). There was a striking similarity between the fractional rate for deconjugation alone and that for deconjugation plus dehydroxylation for both bile acids in individual samples (r = 0.999, p less than 0.001), suggesting that these two processes might occur simultaneously and probably involve the same bacteria. We conclude that our scintiscanning technique provides an accurate, noninvasive method of measuring fractional turnover rate of a bile acid in humans, and that the finding that 75SeHCAT remains conjugated with taurine during enterohepatic recycling means that absorption should be specific for the ileal active transport site, thus rendering it an ideal substance for assessing ileal function.