Bile acids with a cyclopropyl-containing side chain. 1. Preparation and properties of 3 alpha, 7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid

3 alpha, 7 beta-Dihydroxy-22, 23-methylene-5 beta-cholan-24-oic acid (5), a side-chain cyclopropyl analogue of ursodeoxycholic acid (1b), has been prepared by the cyclopropanation of 3 alpha, 7 beta-diacetoxy-24-nor-5 beta-chol-22-ene (2) with ethyl diazoacetate , followed by saponification of the resulting cyclopropyl ester (3). The new bile acid presents similar properties to 1b in water. The sodium salt self-aggregates to form micelles at a concentration of 15.5 mM. The cyclopropane ring does not modify the pKa with respect to compound 1b. Cyclopropyl acid 5 is taken up efficiently by rat liver and promptly secreted into bile. It is partially (70%) conjugated with taurine. The bile flow and bile acids and phospholipids secretion are stimulated by 5, while the cholesterol secretion is stimulated by 5 to a lesser extent.     

Pharmacokinetics of ursodeoxycholic acid in rat

The pharmacokinetic behaviour and metabolism of ursodeoxycholic acid (UDCA) have been studied in the rat. After oral administration of both 3H-labelled (4 muCi/kg body wt) and unlabelled (20 mg) UDCA, UDCA appeared in serum almost entirely in conjugated form (taurine conjugated); UDCA was present in bile mostly as taurine conjugated; the more relevant metabolite is 3 alpha,6 alpha, 7 beta-trihydroxycholanoic acid which represents 10% of the total bile acid pool. UDCA increased bile flow and selectively decreased biliary cholesterol secretion, while phospholipid secretion was unaffected. Faecal UDCA excretion was 15-20% while the urinary extraction was 1.5% during 24 h. The data show that UDCA, when administered in high dose, is promptly secreted into bile almost entirely metabolized to tauroursodeoxycholic acid, where it (1) desaturates the cholesterol in bile, (2) exerts choleretic properties.     

Hepatic uptake and biliary secretion of bile acids in the perfused rat liver.

Hepatic uptake and biliary secretion have been evaluated in the isolated perfused rat liver for cholic, chenodeoxycholic, ursodeoxycholic acid, both free and taurine-conjugated; the physicochemical properties of the bile acids have also been calculated and related to these experimental parameters. Cholic acid disappearance rate from the perfusate was the fastest, followed by that of ursodeoxycholic and chenodeoxycholic; it was also faster for taurine-conjugated bile acids than for their respective unconjugated forms. The recovery in bile was higher for conjugated than for unconjugated bile acids, and among each class, was higher for cholic than for chenodeoxycholic and ursodeoxycholic. The hepatic uptake correlated negatively (r = -0.99) with the bile acid lipophilicity, while the biliary secretion correlated with the solubility of the molecules. These results show the effect of the physicochemical properties of BA on their hepatic handling, at the physiological concentration of BA in the portal blood.     

Comparison of the choleretic properties of bile acids

The choleretic properties of cholic, chenodeoxycholic, and deoxycholic acid and their taurine and glycine conjugates were compared to their ability to form micelles. It has previously been concluded that deoxycholate has the lowest critical micellar concentration; chenodeoxycholate is slightly higher and cholic is much higher. Conjugation with glycine and taurine has little or no effect on the critical micelle concentration. Since the choleretic properties of bile salts are thought to be directly proportional to their osmotic activities, one might suspect that deoxycholic acid would be the least choleretic, chenodeoxycholic slightly more choleretic and cholic much more choleretic, with little difference between the conjugated and unconjugated forms. However, in the present study, cholic, chenodeoxycholic and taurocholic acid produced similar increases in bile flow (450–700 μl/kg) after an equimolar dose (55 μM/kg). Except for the conjugation of deoxycholic acid with taurine, conjugation of these bile acids with glycine or taurine always decreased the choleretic properties of the bile acids. Therefore, it has been concluded that there is not a good correlation between the in vitro osmotic properties of bile acids and their ability to increase bile flow.     

Bile acid conjugation in the chimpanzee: effective sulfation of lithocholic acid

To characterize the hepatic biotransformation in the chimpanzee of the primary bile acid chenodeoxycholic acid (chenic) and its major bacterial metabolite lithocholic acid (lithocholic) a mixture of tracer amounts of¹⁴C-lithocholic and³H-chenic was injected intravenously into two animals with a bile fistula; the chemical form of radioactivity appearing in bile was inferred using thin layer chromatography. About 80% of chenic, and 70% of lithocholic was recovered in 90 min. Chenic was completely conjugated in bile, appearing predominantly as chenyltaurine (52%) and chenylglycine (37%). An unidentified conjugate (about 11%) was also found. Lithocholic was excreted completely as taurine and glycine conjugates, but the majority (63%) of conjugates was sulfated. Sulfation increased progressively with time, and lithocholylglycine was sulfated more than lithocholyltaurine. We conclude that the chimpanzee is similar to man in that the secondary bile acid lithocholic is efficiently sulfated. The chimpanzee thus differs from the baboon and rhesus monkey which sulfate lithocholic poorly. However, the chimpanzee differs from man and is similar to the baboon and rhesus monkey in showing preferential conjugation of bile acids with taurine. The results imply that hepatotoxicity caused by chenic, which is well documented in the rhesus monkey and baboon and has been related to defective lithocholic sulfation, should not occur in the chimpanzee.The work was supported by GSF, as well as Mayo Foundation, Alexander von Humboldt Foundation and Grant AM 16770     

2-Fluoro-beta-alanine, a previously unrecognized substrate for bile acid coenzyme A:amino acid:N-acyltransferase from human liver

Our laboratory has demonstrated recently that conjugates of 2-fluoro-beta-alanine (FBAL) and bile acids are the major biliary metabolites of 5-fluorouracil (FUra) in cancer patients. Bile acids are normally conjugated with glycine or taurine, and therefore the identification of the FBAL-bile acid conjugates suggested that FBAL may also be a substrate for the bile acid conjugating enzyme, bile acid CoA:amino acid:N-acyltransferase. Enzyme activity detected using glycine and taurine as substrates was purified 8-fold from human liver cytosol using a DEAE-cellulose column. This preparation when tested for its activity towards beta-alanine and FBAL using cholyl CoA as the bile acid substrate only catalyzed the formation of FBAL-cholate. beta-Alanine was not a substrate. Confirmation of FBAL-cholate as the enzymatic product was demonstrated by (1) coelution of the product of this reaction on HPLC with authentic FBAL-cholate, (2) specific hydrolysis of this product by cholylglycine hydrolase, and (3) molecular weight of the product (497) being identical to that of the authentic FBAL-cholate. Kinetic experiments demonstrated that the enzyme had an affinity for FBAL (Km 1.45 mM) comparable to taurine (Km 1.32 mM), but greater than glycine (Km 6.45 mM). Formation of FBAL-cholate was inhibited competitively by taurine (Ki 1.27 mM) and glycine (Ki 4.47 mM), suggesting that a single enzyme is responsible for conjugation of glycine, taurine and FBAL with bile acids. These data indicate that the formation of the FBAL-bile acid conjugates in patients receiving FUra results from high affinity of the bile acid conjugating enzyme for FBAL.     

3alpha-6alpha-Dihydroxy-7alpha-fluoro-5beta-cholanoate (UPF-680), physicochemical and physiological properties of a new fluorinated bile acid that prevents 17alpha-ethynyl-estradiol-induced cholestasis in rats

3alpha-6alpha-Dihydroxy-7alpha-fluoro-5beta-cholanoate (UPF-680), the 7alpha-fluorine analog of hyodeoxycholic acid (HDCA), was synthesized to improve bioavailability and stability of ursodeoxycholic acid (UDCA). Acute rat biliary fistula and chronic cholestasis induced by 17alpha-ethynyl-estradiol (17EE) models were used to study and compare the effects of UPF-680 (dose range 0.6-6.0 micromol/kg min) with UDCA on bile flow, biliary bicarbonate (HCO(3)(-)), lipid output, biliary bile acid composition, hepatic enzymes and organic anion pumps. In acute infusion, UPF-680 increased bile flow in a dose-related manner, by up to 40.9%. Biliary HCO(3)(-) output was similarly increased. Changes were observed in phospholipid secretion only at the highest doses. Treatment with UDCA and UPF-680 reversed chronic cholestasis induced by 17EE; in this model, UDCA had no effect on bile flow in contrast to UPF-680, which significantly increased bile flow. With acute administration of UPF-680, the biliary bile acid pool became enriched with unconjugated and conjugated UPF-680 (71.7%) at the expense of endogenous cholic acid and muricholic isomers. With chronic administration of UPF-680 or UDCA, the main biliary bile acids were tauro conjugates, but modification of biliary bile acid pool was greater with UPF-680. UPF-680 increased the mRNA for cytochrome P450 7A1 (CYP7A1) and cytochrome P450 8B (CYP8B). Both UDCA and UPF-680 increased the mRNA for Na(+) taurocholate co-transporting polypeptide (NCTP). In conclusion, UPF-680 prevented 17EE-induced cholestasis and enriched the biliary bile acid pool with less detergent and cytotoxic bile acids. This novel fluorinated bile acid may have potential in the treatment of cholestatic liver disease.     

Ionization of unconjugated, glycine- and taurine-conjugated bile acids by electrospray ionization mass spectrometry

We investigated the effect of organic anions as spray liquid additives on the ionization efficiency of unconjugated, glycine-conjugated and taurine-conjugated bile acids under electrospray ionization conditions. Addition of organic acids influenced the ionization efficiency of whole bile acids. Use of a stronger acid reduced the peak intensity of unconjugated and glycine-conjugated bile acids, while the use of TFA, the strongest acid tested, improved the intensity of taurine conjugates. The hydroxyl group at the C-12 position of cholic acid and deoxycholic acid easily underwent intra-molecular hydrogen bonding with the side chain carboxyl group, accelerating the ionization efficiency. This intra-molecular hydrogen bond may also affect the formation of product ions in low energy-CID. The addition of ammonium ions to the spray liquid influenced the ionization of all bile acids, specifically enhancing the ionization efficiency of unconjugated bile acids.     

Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: Role of glycine conjugates

Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24 h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5–4 h) to 5–100 μM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4 h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH.     

INFLUENCE OF BILE ACIDS ON THE BILIARY TRANSPORT MAXIMUM OF PHENOLSULPHONPHTHALEIN IN THE RAT

1. The effect of changes in bile acid secretion induced by cholestyramine treatment or taurocholate infusion on the biliary transport maximum (Tm) of phenolsulfonphthalein (PSP) was studied in Wistar rats. 2. Five hours after oral administration of cholestyramine (1.5 g/kg bodyweight) the biliary output of bile acids decreased to 51% and bile flow to 76% of control values. The percentage of conjugated and unconjugated PSP excreted into bile and the Tm of the dye were not significantly modified by cholestyramine pretreatment. 3. Administration of sodium taurocholate at increasing rates (60-480 nmol/100 g bodyweight per min) enhanced bile flow and the biliary output of bile acids in a linear dose-related fashion. The Tm of PSP increased progressively until a maximum of 29% above the control values was reached at a taurocholate dose of 240 nmol/100 g bodyweight per min). The enhancement corresponded mainly to the unconjugated dye, the excretion of conjugated PSP not being significantly modified by the infusion of the bile acid. 4. The results indicate that bile acids can influence to some extent biliary excretion of PSP in the rat, although this component is of minor importance at low bile acid secretory rates.     

Taurine ameliorates cholesterol metabolism by stimulating bile acid production in high‐cholesterol‐fed rats

This study was designed to investigate the effects of dietary taurine on cholesterol metabolism in high‐cholesterol‐fed rats. Male Sprague‐Dawley rats were randomly divided into two dietary groups (n = 6 in each group): a high‐cholesterol diet containing 0.5% cholesterol and 0.15% sodium cholate, and a high‐cholesterol diet with 5% (w/w) taurine. The experimental diets were given for 2 weeks. Taurine supplementation reduced the serum and hepatic cholesterol levels by 37% and 32%, respectively. Faecal excretion of bile acids was significantly increased in taurine‐treated rats, compared with untreated rats. Biliary bile acid concentrations were also increased by taurine. Taurine supplementation increased taurine‐conjugated bile acids by 61% and decreased glycine‐conjugated bile acids by 53%, resulting in a significant decrease in the glycine/taurine (G/T) ratio. Among the taurine‐conjugated bile acids, cholic acid and deoxycholic acid were significantly increased. In the liver, taurine supplementation increased the mRNA expression and enzymatic activity of hepatic cholesterol 7α‐hydroxylase (CYP7A1), the rate‐limiting enzyme for bile acid synthesis, by three‐ and two‐fold, respectively. Taurine also decreased the enzymatic activity of acyl‐CoA:cholesterol acyltransferase (ACAT) and microsomal triglyceride transfer protein (MTP). These observations suggest that taurine supplementation increases the synthesis and excretion of taurine‐conjugated bile acids and stimulates the catabolism of cholesterol to bile acid by elevating the expression and activity of CYP7A1. This may reduce cholesterol esterification and lipoprotein assembly for very low density lipoprotein (VLDL) secretion, leading to reductions in the serum and hepatic cholesterol levels.     

Urinary metabolites of dodecylcyclohexane in Salmo gairdneri: evidence of aromatization and taurine conjugation in trout

1. The urinary metabolites of 3H-dodecylcyclohexane were investigated in rainbow trout, Salmo gairdneri R. after a single intragastric dose. In 72 h, 14% of the ingested radioactivity was excreted in urine. 2. Cyclohexylacetic acid, 1-hydroxy-, 3-hydroxy- and 4-hydroxy-cyclohexylacetic acids were present in the unconjugated fraction. 3. In the glucuronide fraction (1.2% dose) labelled aglycones were cyclohexylacetic acid and phenylacetic acid. 4. More than 30% of the urinary 3H was present as phenylacetic and cyclohexylacetic acids conjugated with taurine.     

Studies on the metabolism of propafenone. 2nd comm.: studies on the biotransformation in the dog

Following administration of 14C- and 2H-labelled propafenone hydrochloride (2-(2'-hydroxy-3'-propylaminopropoxy)-omega-phenyl-propiophenone hydrochloride) to beagle dogs, the metabolites were isolated from urine and bile and analysed by mass spectrometry. Reference substances were synthesized on the basis of the structures postulated from the mass spectra and compared with the substances isolated from the biological material. Propafenone was absorbed completely following i.d. administration and eliminated mainly with the bile. Within 28 h 10% of the dose was excreted with the urine and 87% with the bile. Propafenone was extensively metabolized. Less than 1% of the dose was recovered as unchanged substance in urine and bile. The urinary and biliary metabolites were almost exclusively conjugated. The main metabolite, accounting for more than 30% of the dose, was propafenone glucuronide, followed by conjugates of hydroxylated propafenone derivatives with glucuronic acid and sulphuric acid. 5-Hydroxy-propafenone, a propafenone derivative hydroxylated in the middle aromatic ring, and a derivative hydroxylated in the omega-phenyl ring each accounted for about 15% of the dose. Besides these monohydroxy metabolites, two other O-methylated catechol-like derivatives, substituted in the different aromatic rings were recovered. The remainder of the metabolic products identified were mainly substances resulting from degradation of the propoxyamine side chain.     

Bile acids with a cyclopropyl-containing side chain. 3. Separation, identification, and properties of all four stereoisomers of 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid

3 alpha,7 beta-Dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid (CUDCA) (2a), a side-chain cyclopropylog of ursodeoxycholic acid (UDCA) was shown to be a mixture of four stereoisomers (CUDCA A-D). The 22S,23R, and 22R,23S diastereoisomers have been separated, their respective configurations assigned by 13C NMR spectroscopy, and original synthetic schemes for their preparation elaborated. Moreover, theoretical models of the structure of UDCA and CUDCA A-D were built by using molecular computer graphic techniques. It was shown that the four diastereoisomers greatly differ in hydrophilicity, in critical micellar concentration (CMC) in water, and exhibit a different interaction with intestinal bacterial enzymes. It was also shown that CUDCA A-C are not conjugated with glycine or taurine in the liver, while CUDCA D is secreted into bile predominantly as taurine and glycine conjugate.     

Bile salt- and lysophosphatidylcholine-induced membrane damage in human erythrocytes.

The interaction of bile salts and lysophosphatidylcholine (LPC) with membranes has implications both in understanding the aetiology of a number of gastrointestinal disorders, including gastritis, gastric ulcers and colonic cancer, and in enhancing drug absorption by various epithelia. The membrane toxicity of nine bile salts (the sodium (S) salts of chenodeoxycholate (CDC), deoxycholate (DC) and cholate (C) and their glycine (G) and taurine (T) conjugates) and LPC was determined using erythrocyte haemolysis as a model parameter. Washed human erythrocytes were incubated for 15-60 min at 20 degrees C with media buffered at pH 8, 7 and 6. Bile salt toxicity was shown to be a function of type, concentration, pH and contact time with the membrane. At pH 7 toxicity decreased in the order LPC greater than unconjugated dihydroxy salts (SDC and SCDC) greater than conjugated deoxycholates (SGDC and STDC) greater than conjugated chenodeoxycholates (SGCDC and STCDC) greater than unconjugated trihydroxy salt (SC) greater than conjugated trihydroxy salts (SGC and STC). Incubation with equimolar combinations of bile salts (SDC+SCDC; STCDC+SGDC; SDC+STDC) indicated that the resultant damage was an additive function of the damage induced by the individual bile salts.     

Interaction of fusidates with bile acid uptake by isolated rat hepatocytes

Summary The interaction of fusidic acid and two of its conjugates with carrier-mediated uptake of bile acids was investigated in isolated rat hepatocytes. All three fusidates inhibited the uptake of both cholate and taurocholate competitively suggesting a direct interaction of fusidates with bile acid carrier. The inhibition constants for all three fusidates for the inhibition of cholate uptake were significantly different from the respective inhibition constants for the inhibition of taurocholate uptake. This would indicate that both cholate and taurocholate are transported by more than one carrier into hepatocytes. The results may also indicate that taurine conjugated bile acids may be transported preferentially by one transport system while unconjugated bile acids may be preferentially transported by another transport system.Part of this publication was presented in the 18. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft (1977)     

Studies on the Metabolism of Arylacetic Acids: 5. The Metabolic Fate of 2-Naphthylacetic Acid in the Rat,Rabbit and Ferret

1. The fate of [¹⁴C]-2-naphthylacetic acid has been studied in rat, ferret and rabbit and the findings compared with those for the 1-isomer.

2. 2-Naphthylacetic acid was conjugated in all three species with glycine, glutamine, taurine and glucuronic acid.

3. In rat, but not rabbit, 2-naphthylacetic acid is eliminated in the bile (50% dose) and undergoes entergohepatic circulation.

4. The pattern of metabolism and elimination of 2-naphthylacetic acid in rat is dose-dependent. Small doses (5 mg/kg) are excreted in urine and bile mainly as the glucuronic acid conjugate, while larger doses (250 mg/kg) are eliminated in the bile mainly as amino acid conjugates.

5. Metabolic conjugation of 1- and 2-naphthylacetic acids differs in the three species studied. 1-Naphthylacetic acid is conjugated largely with glucuronic acid and amino acid conjugation is minimal, whereas the converse is true of the 2-isomer. These differences in conjugation have been correlated with structural differences between the two acids.

6. 2-Naphthylacetic acid is the first carboxylic acid found to undergo simultaneous amino acid conjugation with glycine, glutamine and taurine in non-primate species. 2-Naphthylacetic acid may be a useful probe for exploring the species occurrence of the various conjugations.     

Basic cholane derivatives. XI: Comparison between acid and basic derivatives.

A series of hydroxycholan-24-amines was synthesized; the carboxyl group of starting unconjugated bile acids was transformed into a basic moiety [-NH2, -NHCH3, -N(CH3)2, or -NHCH2C6H5] at C-24. Solubilities, acidities, partition coefficients, and critical micellar concentrations were measured and compared with those of the corresponding bile acids. Because the steroid nucleus in the amines is the same as that in the bile acids, most of the physical-chemical properties of the two compound classes were similar. The amines were more soluble than the corresponding acids; solubilities depended mainly on the number of steroid hydroxyls and, to a lesser extent, on the side chain. Amines are strong bases in water, whereas unconjugated bile acids can be classified as weak acids. N-Benzylamino derivatives have higher log P (P is partition coefficient) values, as a consequence of the bulky hydrophobic substituent; the log P values were almost the same for the amines and the bile acids and depended on the steroid hydroxyls. Amines can self-aggregate at an acidic pH and form cationic micelles; the critical micellar concentrations of amines were of the same order of magnitude as those of bile acids. The introduction of a basic function in the side chain of the cholane moiety increased the antimicrobial activity toward most gram-positive strains.     

Bile acids in porcine fetal bile

A study of the biliary bile acid composition in porcine fetus compared with that of the adult pig is described. Biles, collected during gestation (weeks 4, 15 to 17 and at birth), aged six months and two years old, were analyzed by gas-liquid chromatography and capillary GC-MS. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, piperidinohydroxypropyl Sephadex LH-20. All and one fourth of the total bile acids in the bile of weeks 4 and 15 of gestation, respectively, were present as unconjugated form, however, only a trace of unconjugated bile acids was present in bile of late gestation, the young and the adult pigs. The ratio of glycine/taurine (G/T) conjugates in the conjugated fraction of the fetal bile at 15 weeks gestation was less than 1, which markedly contrasted with the conjugation pattern for adult bile where the ratio of G/T conjugates was approximately more than 9. The predominant acids identified in porcine fetal bile of the 4 weeks gestation were cholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-chola n-24-oic acid) and chenodeoxycholic acid (3alpha,7alpha -dihydroxy-5beta-cholan-24-oic acid). However, cholic acid in late gestation, young, and adult bile was the smallest component, whereas chenodeoxycholic acid was still the major constituent of these biles. The presence of small but valuable amounts of allocholic acid (3alpha,7alpha,12alpha-trihydroxy-5alpha-chol an-24-oic acid) and cholic acid in early gestation suggested the presence of 12alpha-hydroxylase activity of steroid nucleus in fetal liver. Considerable amounts of glycine-conjugated hyodeoxycholic acid were found in the bile of the gestation periods, suggesting the placental transfer of this bile acid from maternal circulation.     

Enterohepatic circulation of T-2 toxin metabolites in the rat

The enterohepatic circulation of T-2 toxin and its conjugated metabolites was examined in bile duct-cannulated male rats. Rats administered tritiated T-2 toxin intraduodenally (id) eliminated 44.65% and 57.25% of the administered dose in the bile within 4 and 8 hr post-dosing, respectively. TLC profiles of the T-2 metabolites were similar after intravascular and id administration. The major metabolites detected were 3'-OH-hydroxytryptamine-2 (HT-2), glucuronic acid conjugates, T-2 tetraol (TOL), 4-deacetylneosolaniol (4-DN), and HT-2. Tritium-labeled glucuronides obtained from the bile of rats administered [3H]T-2 toxin intravascularly were extracted and purified using C-18 and silica column chromatography. Enzymatic hydrolysis followed by TLC and GC/MS indicated that the aglycone portion of the glucuronides were composed of 3'-OH HT-2, HT-2, 4-DN, and TOL. After id administration of the glucuronides the rats eliminated 6.01% (4 hr) and 11.86% (8 hr) of the dose in the bile. No free metabolites of T-2 toxin were detected in the bile of any animals administered the purified glucuronides. Oral treatment of the rats with the beta-glucuronidase inhibitor, saccharolactone, did not produce a significant decline in the amount of radioactivity recovered in the bile following administration of the tritium-labeled glucuronides. These studies substantiate the enterohepatic circulation of T-2 toxin metabolites.