Fluorescent choleretic and cholestatic bile salts take different paths across the hepatocyte: transcytosis of glycolithocholate leads to an extensive redistribution of annexin II.

We have used fluorescent derivatives of the choleretic bile salts cholate and chenodeoxycholate, the cholestatic salt lithocholate, and the therapeutic agent ursodeoxycholate to visualize distinct routes of transport across the hepatocyte and delivery to the canalicular vacuole of isolated hepatocyte couplets. The cholate and chenodeoxycholate derivatives produced homogeneous intracellular fluorescence and were rapidly transported to the vacuole, while the lithocholate analogue accumulated more slowly in the canalicular vacuole and gave rise to punctate fluorescence within the cell. Fluorescent ursodeoxycholate showed punctate intracellular fluorescence against a high uniform background indicating use of both pathways. Inhibition of vesicular transport by treatment with colchicine and Brefeldin A had no effect on the uptake of any of the compounds used, but it dramatically impaired delivery of both the lithocholate and the ursodeoxycholate derivatives to the canalicular vacuole. We conclude that while the chenodeoxycholate and cholate analogues traverse the hepatocyte by a cytoplasmic route, lithocholate and ursodeoxycholate analogues are transported by vesicle-mediated transcytosis. Treatment of couplets with glycine derivatives of lithocholate and ursodeoxycholate, but not cholate or chenodeoxycholate, led to a marked relocalization of annexin II, which initially became concentrated at the basolateral membrane, then moved to a perinuclear distribution and finally to the apical membrane as the incubation progressed. This suggests that lithocholate and ursodeoxycholate treatment leads to a rapid induction of transcytosis and that annexin II exchange occurs upon membrane fusion at all stages of the hepatocyte transcytotic pathway. These results indicate that isolated hepatocyte couplets may provide an inducible model system for the study of vesicle-mediated transcytosis.

Biliary excretion of chenodeoxycholyllysylrhodamine in Wistar rats: a possible role of a bile acid as a carrier for drugs.

The effect on biliary excretion of rhodamine after its conjugation to give chenodeoxycholyl-lysyl-rhodamine (cheno-lys-R) was studied in male Wistar rats. Following its intravenous injection via the jugular vein of animals cheno-lys-R was efficiently excreted into bile with a peak biliary excretion of 31.6 +/- 1.2% dose 5 min-1 and a cumulative biliary excretion of 96.4 +/- 2.0% in 30 min of the total dose administered. Unlike cheno-lys-R, rhodamine had a poor biliary excretion of 1.0 +/- 0.1% dose 5 min-1 and a cumulative biliary excretion of 3.3 +/- 0.6% in 30 min. Cheno-lys-R had a short plasma half-life (t1/2 alpha) of 4.0 +/- 0.5 min, whereas free rhodamine had a longer half life (t1/2 alpha) of 82.1 +/- 1.4 min. The plasma clearances of cheno-lys-R and rhodamine were 41.2 +/- 6.5 and 9.0 +/- 1.2 ml/min per kg, respectively. The data indicate that the cationic fluorescent xenobiotic, rhodamine, when conjugated to the bile salt analogue, greatly increased the biliary excretion of rhodamine and that cheno-lys acted as a carrier for hepatic uptake of rhodamine. Thus, an appropriate bile salt derivative may be used to target a drug to the liver.

Ileal absorption of tyrosine-conjugated bile acids in Wistar rats.

We recently reported that tyrosine-conjugated bile acids, when injected intravenously into bile-fistula rats, are extracted by the liver and secreted intact into bile with an efficiency similar to that seen for taurocholate. Now the effect of tyrosine and glycyltyrosine conjugation of bile acids on ileal absorption has been studied in Wistar rats. 125I-labelled tyrosine- and glycyltyrosine-conjugated bile acid or [14C]taurocholate was injected in 400 microliters aliquots of physiological saline buffered to pH 7.8 into the ileal lumen of bile-fistula rats. Recovery of bile salts in bile was taken as proof of ileal absorption. In comparison with taurocholate, ileal absorption was about 10% less for cholyltyrosine and chenodeoxycholyltyrosine and about 50% less for deoxycholyltyrosine. Thus, tyrosine-conjugated bile acids are absorbed by the ileum and excreted into bile and may undergo enterohepatic circulation. Low recoveries of deoxycholyltyrosine relative to deoxycholylglycine suggested that side chain structure was important for ileal absorption of 3 alpha,12 alpha-dihydroxy bile acids. Elongation of cholic acid to form cholylglycyltyrosine markedly reduced 90-min cumulative ileal absorption relative to cholyltyrosine. Although initial rates of recovery of cholylglycyltyrosine were comparable to those of the other bile acids, very little further absorption was seen in the last hour of the experiment, suggesting that this compound was rapidly degraded within the intestinal lumen.

Synthesis and biliary excretion of tyrosine-conjugated bile salts in Wistar rats.

Tyrosine-labelled free and glycine-conjugated bile acids were synthesized and radiolabelled with 125I to high purity. The synthetic method utilized excess tyrosine methyl ester hydrochloride (1.4 equiv.) and bile acid (one equiv.) via dicyclohexylcarbodiimide (1.4 equiv.) with yields of 90-93% for tyrosine bile acid conjugates and glycyltyrosine conjugates and 56-60% yields for the glycylglycyltyrosine conjugates. All of the eight iodinated tyrosine bile acids tested were rapidly excreted into bile following intravenous injection. In bile duct-cannulated rats with ligated renal pedicles under pentobarbital anaesthesia the percentages of injected dose recovered from bile within 20 min were as follows: cholylglycine ([14C]cholylGly), 81.2 +/- 1.3%; taurocholate ([14C]taurocholate), 94.3 +/- 1.0%; cholyltyrosine (125I-cholylTyr), 85.5 +/- 3.3%; deoxycholyltyrosine (125I-deoxycholylTyr), 87.9 +/- 6.3%; chenodeoxycholyltyrosine (125I-chenodeoxycholylTyr), 93.4 +/- 2.9; cholylglycyltyrosine (125I-cholylGlyTyr), 95.7 +/- 6.7%; deoxycholylglycyltyrosine (125I-deoxylcholylGlyTyr), 92.5 +/- 3.2%; chenodeoxycholylglycyltyrosine (125I-chenodeoxycholylGlyTyr), 94.1 +/- 3.1%; cholyldiglycyltyrosine (125I-cholylGlyGlyTyr), 85.2 +/- 3.6%, and deoxycholyldiglycyltyrosine (125I-deoxycholylGlyGlyTyr), 85.5 +/- 2.7%. Values are means +/- SD. Thus the biliary excretion of 125I-chenodeoxycholylGlyTyr, 125I-chenodeoxycholylTyr, 125I-deoxycholylGlyTyr and 125I-cholylGlyTyr was similar to that of [14C]taurocholate, the major naturally occurring bile acid in the rat, and the biliary excretion of all the tyrosine conjugates was similar to or exceeded that of [14C]cholylglycine. Conjugation with tyrosine enhanced the efficiency of plasma-to-bile transport of most naturally occurring bile acids. Comparison of glycyltyrosine conjugates with glycylglycyltyrosine conjugates suggests that any additional benefit derived by elongation of the side-chain is probably negated by obscuring the 12 alpha-hydroxyl function on the steroid nucleus in the bile acid glycylglycyltyrosine conjugates.

The effect of tyrosine conjugation on the critical micellar concentration of free and glycine-conjugated bile salts.

We investigated the effect of conjugation with the aromatic amino acid tyrosine on the critical micellar concentration (CMC) of bile salts. The CMC values were determined by surface tension and by dye solubilization. The surface tension measurement employed the Du Nouy ring detachment method and the dye solubilization measurement utilized a water-insoluble dye, 1-O-tolylazo-2-naphthol. We compared the CMC values of the sodium salts of cholyltyrosine (cholylTyr), deoxycholyltyrosine (deoxycholylTyr), deoxycholylglycyltyrosine (deoxycholylGlyTyr) chenodeoxycholyltyrosine (chenodeoxycholylTyr), chenodeoxycholylglycyltyrosine (chenodeoxycholylGlyTyr), cholyldiglycyltyrosine (cholylGlyGlyTyr) and cholylglycyltyrosine (cholylGlyTyr) with their respective glycine conjugated bile salts. Both techniques of CMC determination indicated that tyrosine conjugation to free and glycine-conjugated bile salts reduced their CMC significantly.

The effect of bile salts on human vascular endothelial cells.

The uptake and release of radiochromium from adult human vascular endothelial cells in culture was employed to determine the relative toxicity of different bile salts. Endothelial cells after pre-incubation with 51Cr for 18 h were incubated with bile salts for 24 h and percentage chromium release was taken as a measure of toxicity to cells. Lithocholic acid (LC) (potassium salt) was cytotoxic at concentrations greater than 50 microM. However, LC glucuronide, sulfate and the beta-epimer were progressively less toxic with toxicity seen at concentrations of 60, 110 and 180 microM, respectively. The greatest cytotoxic effect was observed with glycolithocholic acid (GLC) (potassium salt) which was toxic at every concentration tested (20-200 microM). Sulfation abolished the toxic effect of GLC. At the concentrations employed for the assay (between 20 and 240 microM) GLC sulfate (disodium salt), taurolithocholic acid sulfate (disodium salt), cholic acid (sodium salt), glycocholic acid (sodium salt), deoxycholic acid (sodium salt) and ursodeoxycholic acid (sodium salt) were not cytotoxic. The 51Cr release cytotoxicity assay was validated with lactate dehydrogenase leakage from endothelial cells with a good correlation (r = 0.87). These data confirm in a human cellular system that LC and its conjugates were the most toxic of the bile salts tested and explains its pathophysiological importance in hepatobiliary disease. It also suggests that biotransformation by either sulfation or beta-epimerisation of bile salts especially of LC, as occurs in patients with intrahepatic or extrahepatic biliary obstruction or severe cholestasis, is hepatoprotective.

Cholyl-lysylfluorescein: synthesis, biliary excretion in vivo and during single-pass perfusion of isolated perfused rat liver.

A fluorescent bile salt, cholyl-lysylfluorescein (cholyl-lys-F), was synthesised so that it retained both an intact steroid ring and a side chain structure with an unblocked carboxyl group. Its biliary kinetics and hepatic extraction were studied in Wistar rats and in the isolated perfused rat liver, respectively. The synthetic method used excess N-epsilon-CBZ-l-lysine methyl ester hydrochloride (7 mmol) and cholic acid (5 mmol) via EEDQ with a yield of 94% for cholyl-lys. Cholyl-lys-F was synthesized employing equimolar amounts of cholyl-lys (sodium salt) and fluorescein isothiocyanate (FITC) in bicarbonate buffer (pH 9.5) over 16 h at room temperature (21 degrees C) with a yield of 70%. The fluorescent property of cholyl-lys-F was similar to fluorescein with a strong apple-green fluorescence. In bile-fistula rats under pentobarbital anaesthesia, the cumulative 20 min biliary excretion as a percentage of injected dose were as follows: cholyl-lys-F, 94.4 +/- 0.3%, [14C]cholylglycine (CG), 93.1 +/- 1.2% and fluorescein (F), 34.8 +/- 0.5. Furthermore the single-pass hepatic extraction of cholyl-lys-F was 64.1 +/- 3.9%, [14C]CG was 66.1 +/- 1.2% and F was 16.5 +/- 2%. The similarity in biliary output and hepatic extraction of cholyl-lys-F to that of the natural bile acid cholylglycine suggest that both compounds are handled in a similar fashion. The greater biliary excretion and hepatic extraction of cholyl-lys-F relative to free fluorescein further suggest that conjugation with a bile salt may be an efficient way of targeting compounds to the liver.

Biliary lipid output by isolated perfused rat livers in response to cholyl-lysylfluorescein.

The biliary output of bile acids and lipids is tightly coupled. The ability of the natural bile acid glycocholate to trigger biliary lipid secretion was compared with that of the fluorescent bile acid analogue cholyl-lysylfluorescein (cholyl-lys-F). When administered as a 5 min pulse of 2.5 mumol/min to bile acid-depleted rat livers perfused under recycling conditions, glycocholate produced well-defined peaks of phospholipid and cholesterol output, and of bile flow, which were coincident with the peak of bile acid output. Although cholyl-lys-F did trigger biliary lipid secretion, its time course of appearance was delayed and well-defined peaks of output were not observed. However, the increased biliary output of phospholipid and cholesterol was coincident with that of bile acids and, as judged by phospholipid/bile acid and cholesterol/bile acid ratios, cholyl-lys-F was as effective as glycocholate in triggering biliary lipid output. When administered to livers perfused under single pass conditions, perfusate to bile transfer of glycocholate was > 85% at infusion rates of up to 5 mumol/min whereas transfer of cholyl-lys-F showed saturation at infusion rates of > 0.2 mumol/min; the time course of biliary output of both bile acids was similar. Thus, under recycling conditions, cholyl-lys-F not taken up during first pass will be continually represented for transfer to bile, explaining why bile acid and lipid output did not occur as well-defined peaks.

Synthesis, physical and biological properties of lithocholyl-lysyl-fluorescein: a fluorescent monohydroxy bile salt analogue with cholestatic properties.

We have synthesised and characterised a fluorescent monohydroxy bile salt analogue, lithocholyl-lysyl-fluorescein and compared its physical and biological properties with those of lithocholate, glycolithocholate, sulpholithocholate, lithocholic acid glucuronide and taurocholate. The synthetic method used excess N-epsilon-CBZ-L-lysine methyl ester hydrochloride and lithocholic acid via N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinolone (EEDQ) to give lithocholyl-lysine. Lithocholyl-lysyl-fluorescein (LLF) was then prepared using equimolar amounts of lithocholyl-lysine and fluorescein isothiocyanate (FITC) in bicarbonate buffer. LLF retained an apple green fluorescence, similar to that of fluorescein. Unlike lithocholate, the critical micellar concentrations (CMCs) of LLF, glycolithocholate (GLC), lithocholic acid glucuronide (LG) and sulpholithocholic acid (SLC) were similar. HPLC retention times (tRs) of LLF and GLC were similar with a ratio of LLF/GLC of 1.05. In contrast, the tR of SLC (6.52 min) but not of LG (21.2 min) was more comparable to that of taurocholate (5.73 min). In rats under pentobarbital anaesthesia, the plasma half-life (t(1/2alpha)) (min) was 4.5 +/- 1.3 (n = 6) for LLF, 2.9 +/- 0.4 (n = 5) for [14C]sulpholithocholate (14C-SLC) and 4.3 +/- 0.3 (min) for [14C]lithocholic acid glucuronide (14C-LG). Plasma clearances of 14C-SLC, LLF and 14C-LG were 15.5 +/- 2.2 (n = 6), 18.1 +/- 4.2 (n = 6) and 17.8 +/- 0.5 ml/min/kg (n = 6) (P = 0.15), respectively. Biliary excretion in bile-fistula rats gave cumulative 20 min biliary output as a percentage of injected dose as follows: LLF, 71.6 +/- 0.8% (n = 10); 14C-SLC, 75.5 +/- 2.8% (n = 6) and 14C-LG, 61.7 +/- 0.5% (n = 6) (P = NS). Peak biliary excretion rates, given as % dose/2 min, were 10.2 +/- 0.3 for LLF, 13.5 +/- 0.6 for 14C-SLC and 12.8 +/- 0.4 for 14C-LG. In another group of bile-fistula rats, a 3.0 micromol/500 microl saline i.v. bolus of LLF caused a 15.4 +/- 1.9% decrease in bile flow and, similarly, sodium lithocholate in a solution of albumin caused a 17.9 +/- 1.8% (P = NS) diminution in bile flow. Despite the similar cholestatic properties of LLF and lithocholate, LLF was more soluble than lithocholate, with a relative retention time on HPLC similar to that of GLC. LLF is a divalent 'unipolar' anionic fluorescent monohydroxy bile salt analogue with physical, biological and cholestatic properties that are similar to those of lithocholate, glycolithocholate and their derivatives and thus offers a potentially useful probe for studying mechanisms of monohydroxy bile salt-induced cholestasis at the hepatocellular level.

A 51Cr release cytotoxicity assay for use with human intrahepatic biliary epithelial cells.

Human intrahepatic biliary epithelial cells are important immune targets in a variety of hepatobiliary diseases particularly primary biliary cirrhosis and primary sclerosing cholangitis. The ability to isolate and maintain these cells in short term primary tissue culture has permitted us to develop an in vitro cytotoxicity assay for the study of these cells as potential targets to a variety of toxic stimuli. We have therefore established a chromium-51 (51Cr) release cytotoxicity assay for use with primary cultures of human intrahepatic biliary epithelial cells. The method is simple, reproducible and is more sensitive than dye exclusion, light microscopy and release of lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase.

Biliary excretion of fluorescent cholephiles in hepatocyte couplets: An in vitro model for hepatobiliary and hepatotoxicity studies.

Hepatocyte couplets (26.8% of cell preparation with >85% viability) were prepared from rat livers. The preparation maintained the activity of 7-ethoxycoumarin-O-deethylase throughout an 8-hr incubation. Couplets extensively secreted the fluorescent cholephiles, fluorescein and cholyl-lysyl-fluorescein isothiocyanate, into sealed canalicular spaces within 2 hr and 15-30 min, respectively, which is indicative of functional tight junctions. The time scale for biliary secretion of the cholephiles correlated well with the relative kinetics of biliary excretion reported in vivo. Hepatocyte couplets may prove useful for studies on toxicity directed towards the hepatobiliary system.

Cholyllysyl fluroscein and related lysyl fluorescein conjugated bile acid analogues.

There have been attempts to couple bile acids to fluorescein to permit their visualization during studies of physiology and pathophysiology. Although conjugation has been achieved by many, the product differed in many respects from the parent bile acid congener. We describe lysylfluorescein conjugated bile acid analogues (LFCBAA) synthesized in our laboratory as model divalent "unipolar" molecules. We have determined LFCBAA properties including their water:octanol partition coefficient, HPLC retention time and critical micellar concentration and compared them with their parent bile acid congeners. Cholyl lysylfluorescein (CLF) and lithocholyl lysylfluoroscein (LLF) have properties similar to cholylglycine (CG) and glycolithocholate (GLC), respectively. In human and rat hepatocytes uptake of CLF follows Michaelis-Menten kinetics with K(m) and Vmax similar to CG. Biliary excretion rates of CLF and LLF closely resemble those of CG and GLC in both normal and mutant TR- rats which lack the multiorganic anion transporter (MOAT), strongly supporting the notion that CLF and LLF are substrates for the canalicular bile salt transporter (cBST). The close similarity of hepatocyte uptake and biliary secretion of these LFCBAA and their parent bile acid congeners makes them potentially useful probes for the intracellular visualization of bile salt movement and deposition in various models of bile formation and secretion.

Selectively reduced biliary excretion of cholyldiglycylhistamine but not of cholyltetraglycylhistamine in ethinyl estradiol-treated rats. A possible indicator of increased bile canalicular permeability.

A series of bile acid derivatives were synthesized, purified and radiolabelled. These were [125I]cholylglycylhistamine [( 125I]CGH), [125I] cholyldiglycylhistamine [( 125I]CG2H), [125I]cholyltriglycylhistamine [( 125I]CG3H), and [125I]cholyltetraglycylhistamine [( 125I]CG4H). These derivatives were rapidly excreted unchanged into the bile of bile-fistula rats. In normal rats the 30-min cumulative excretion following intravenous administration was only 39.0 +/- 0.7% for [125I]CGH but greater than 80% for the three larger compounds. This marked difference in biliary recovery between CGH and the other larger compounds could be due to a threshold biliary permeability, and we postulated that the critical molecular weight threshold for effective biliary retention of such compounds falls between [125I]CGH (MW 683) and [125I]CG2H (MW 740). Increased permeability, involving a shift to a higher molecular weight threshold would then be anticipated to diminish biliary excretion of [125I]CG2H (MW 740) before exerting a major influence on the biliary excretion of [125I]CG4H (MW 854). We previously reported functional and morphological studies which suggest that ethinyl estradiol (EE) may alter the permeability of bile canalicular tight junctions. In this study we have looked for further evidence of a progressive permeability change in EE-induced cholestasis by observing the biliary excretion of CG2H and CG4H in rats. Treatment with EE (5 mg/kg/day) for 3 days (EE3) or with the injection vehicle propylene glycol for 7 days (C7) reduced biliary excretion to a significant extent when compared to 3-day controls (C3) but had no differential effect on the 30-min recoveries from bile of CG2H and CG4H, respectively: C3 (81.2 +/- 1.8% and 81.7 +/- 2.1%, P = CN): C7 (72.3 +/- 3.0% and 73.5 +/- 3.6%, P = NS): EE3 61.8 +/- 2.5% and 61.9 +/- 2.7%, P = NS). However, treatment with EE for 7 days significantly reduced the biliary recovery of CG2H (46.8 +/- 9%) compared to EE3 rats (P less than 0.0025) but there was no significant change of biliary CG4H recovery (61.0 +/- 2.5%, P = NS) compared with EE3 rats. These results are compatible with our hypothesis that EE-induced cholestasis is associated with a change of biliary permeability which, as it progresses, affects successively larger molecules.

Biliary permeability during ethinyl estradiol-induced cholestasis studied by segmented retrograde intrabiliary injections in rats.

The influence of ethinyl estradiol (EE) treatment on the permeability of the biliary tree in rats has been assessed by the segmented retrograde intrabiliary injection (SRII) technique. Three pairs of compounds were studied, inert [14C]sucrose and [3H]inulin; [14C]taurocholic acid (TC) and [14C]glycocholic acid (GC) and the non-ionic bile acid derivatives [125I]cholyldiglycylhistamine (CG2H) and [131I]cholyltetraglycylhistamine (CG4H). In control rats recovery in bile after SRII was always greater for the larger of any pair of compounds, confirming that the biliary tree acts as a filter, and that decreased recovery from bile during this technique is an index of greater biliary permeability. After EE treatment recovery of all compounds was significantly reduced, thus confirming that EE increases biliary permeability. Recovery of sucrose and inulin fell from 55-65% of the administered dose in controls to 8-9% in EE rats. Recoveries of TC, GC, CG2H and CG4H was also reduced, but their biliary recovery profiles were consistent with marked re-excretion into bile of that portion which had initially passed out of the biliary system by filtration. During the later phase of the experiment excretion of the negatively charged bile acids TC and GC was greater than that of the non-ionic bile acid derivatives CG2H and CG4H. Although the site at which these permeability changes have occurred is unknown, our results are compatible with previous data implicating increased tight junction permeability as a mechanism of EE-induced cholestasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface tension properties of human urine: relationship with bile salt concentration.

The surface tension of urine has been simply and rapidly measured using two methods, the DuNouy ring detachment method and the Wilhelmy blade immersion method. The methods agree, the correlation coefficient, r, was 0.992. The effect of ageing of the surface, of storage, of temperature, pH, dilution and albumin on urine surface tension are described. Volume corrected 24 hour urine surface tensions from 6 volunteers over 12 or more days showed variations between individuals of mean surface tensions of 6 mN/m (S.D. +/- 2.16 mN/m). This suggests individual variation in excretion of surface active agents, probably bile salts. This was confirmed by further studies which showed that: 1. Surface tension of urines and rediluted extracts were not significantly different when amphiphilic and hydrophobic solutes including bile salts were extracted from urine and subsequently rediluted in water of the same volume. 2. Bile salt concentration in urine measured by RIA and by enzymatic spectrofluorometric methods correlated well with urine surface tension, r = -0.91, and r = -0.60. 3. Molecular surface area for urinary surfactant was 79 A2 similar to pure conjugated bile salt solutions calculated from dilution studies. We conclude that the main determinant of urine surface tension is bile salt concentration.

Lack of evidence for vesicle trafficking of fluorescent bile salts in rat hepatocyte couplets.

The role of intracellular vesicles in the movement of bile salts through hepatocytes from blood to bile has not been resolved. To determine whether bile salts are sequestered during transit, rat hepatocyte couplets were incubated with the fluorescent bile salts cholyl-lysyl-fluorescein (CLF) and chenodeoxycholyl-lysyl-fluorescein (CDCLF). Cellular and canalicular fluorescence were measured by confocal scanning fluorescence microscopy; inhomogeneity in intracellular fluorescence was used to evaluate potential sequestering of bile salts. Mean cellular and canalicular fluorescence increased in parallel over 10 min, slightly exceeding (P < 0.05) the degree of increase in intracellular inhomogeneity. The microtubule inhibitor colchicine had no effect on cellular or canalicular fluorescence patterns. In contrast, the nonfluorescent bile salt taurocholate enhanced the recovery of microtubules from cold-induced depolymerization, measured by confocal immunofluorescence of beta-tubulin. Thus no evidence was obtained for intracellular sequestering of bile salts or microtubule-dependent trafficking before canalicular secretion; cellular uptake and distribution occurred in parallel with canalicular secretion. The previously documented dependence of bile salt secretion on intact microtubule function therefore appears to be an indirect rather than a direct consequence of microtubule-dependent events. In particular, enhanced microtubule assembly may play a role in bile salt-induced delivery of bile salt transporters to the canalicular membrane.

Plasma elimination of cholyl-lysyl-fluorescein (CLF): a pilot study in patients with liver cirrhosis.

BACKGROUND: Cholyl-lysyl-fluorescein (CLF) is a fluorescein-labelled bile acid whose biological behaviour closely resembles that of naturally occurring cholyl glycine. AIM: The aim of this study was to analyze the CLF plasma elimination in patients with liver cirrhosis. METHODS: A dose of CLF at 0.02 mg/kg b.w. was administered i.v. in 26 patients with liver cirrhosis and 9 healthy volunteers. Blood samples were collected before injection and then at 10 min intervals over 60 min. Plasma fluorescence was measured by a luminescence spectrometer and residual fluorescence over the time of the study was compared in each group. Routine liver function tests (rLFTs) were performed before each injection. RESULTS: Plasma elimination of CLF was significantly impaired in patients with cirrhosis compared to healthy subjects with p values <0.0001 at each analyzed time point. CLF test showed 100% sensitivity for liver cirrhosis when residual fluorescence was measured 30, 40, 50 and 60 min after injection. Routine LFTs showed 85% sensitivity for bilirubin, 84% for total bile acids, 69% for aspartate aminotransferase 62% for albumin and 50% for alkaline phosphatase. CLF elimination measured 60 min after injection correlated with Child-Pugh score (r=0.3945; p<0.05) and albumin (rs=0.6451; p<0.001). No adverse reaction or side effects of CLF were observed. CONCLUSIONS: CLF test clearly distinguished between the two analyzed groups and was more sensitive than routine liver function tests. The test appears safe, simple to perform and analyze and after validation in larger cohorts of patients may have the potential to become a useful dynamic test of liver function.

Effect of tauroursodeoxycholate and S-adenosyl-L-methionine on 17beta-estradiol glucuronide-induced cholestasis.

BACKGROUND/AIMS: S-adenosyl-L-methionine (SAMe) and tauroursodeoxycholate (TUDC) exert an additive ameliorating effect on taurolithocholate (TLC)-induced cholestasis. The aims were to investigate the protective effect of SAMe on 17beta-estradiol-glucuronide (17betaEG) cholestasis and to find out whether SAMe and TUDC may exert an additive, ameliorating effect. METHODS: Hepatocyte couplet function was assessed by canalicular vacuolar accumulation (cVA) of cholyllysylfluorescein (CLF). Cells were co-treated with 17betaEG and SAMe, TUDC, or both (protection study), or treated with 17betaEG and then with SAMe, TUDC or both (reversion study) before CLF uptake. Couplets were also co-treated with SAMe and dehydroepiandrosterone (DHEA), a competitive substrate for the sulfotransferase involved in 17betaEG detoxification. The effects of 17betaEG, SAMe and TUDC were also examined on intracellular distribution of F-actin. RESULTS: Both SAMe and TUDC significantly protected against, and reversed, 17betaEG-induced cholestasis, but their effects were not additive. DHEA abolished the protective effect of SAMe. 17BetaEG did not affect the uptake of CLF into hepatocytes at the concentrations used, and also, it did not affect the intracellular distribution of F-actin. CONCLUSIONS: 17BetaEG does not affect the uptake of CLF into hepatocytes. SAMe and TUDC protect and reverse 17betaEG-induced cholestasis, but without an additive effect. Protection by SAMe may involve facilitating the sulfation of 17betaEG.

Effect of anaesthetic agents on bile flow and biliary excretion of 131I-cholylglycyltyrosine in the rat.

We have compared in the rat the effects of i.v. anaesthetic agents on bile flow rate and on the biliary excretion of a novel bile acid, 131I-cholylglycyltyrosine (131I-cholylgly.tyr.). Etomidate 1-mg bolus and 2-mg h-1 infusion, Althesin 3-mg bolus and 14.5-mg h-1 infusion and propofol 3.3-mg bolus and 3.3-mg h-1 were given via a tail vein cannula and pentobarbitone 50 mg kg-1 was given by the intraperitoneal route, to groups of six rats. Each animal received only one anaesthetic agent. One hour after cannulation of the common bile duct, 131I-cholylgly.tyr. 5 microCi was injected into the jugular vein and bile was collected every 1 min for 10 min. The mean (SD) percentage cumulative biliary excretion of 131I-cholylgly.tyr. at the end of 10 min was: propofol group 74.1 (5.2)%; Althesin group 82.3 (2.2)%; etomidate group 69.4 (17.6)%; pentobarbitone group 76.4 (3.2)%. Propofol and Althesin were relatively more choleretic, causing bile flow rates twice that produced by pentobarbitone. Only Althesin caused a significant increase in biliary excretion of 131I-cholylgly.tyr. relative to that in rats that received pentobarbitone. Bile flow rates for the respective anaesthetic techniques (microliter min-1/100 g body weight) (mean (SD)) were: propofol group 14.1 (1.8); Althesin group 12.5 (1.7); etomidate 8.5 (1.4); pentobarbitone group 7.3 (1.0). There was a marked metabolic acidosis in all rats except in the propofol group, in which normal acid-base status and oxygenation were observed.

In vitro anti-HIV-1 virucidal activity of tyrosine-conjugated tri- and dihydroxy bile salt derivatives.

The cellular toxicity and anti-human immunodeficiency virus type 1 (HIV-1) virucidal activity of four synthesized tyrosine-conjugated bile salt derivatives with high surfactant activities, namely di-iodo-deoxycholyltyrosine (DIDCT), di-iodo-chenodeoxycholyltyrosine (DICDCT), di-iodo-cholylglycyltyrosine (DICGT) and deoxycholyltyrosine (DCT), were evaluated and compared with either sodium deoxycholate or nonoxynol-9. DIDCT, DICDCT and DCT but not DICGT showed virucidal activity against three different laboratory-adapted strains of HIV-1 (RF, IIIB and MN). All the bile salt derivatives tested excluding DICGT were virucidal at a concentration as low as 10 ng/mL. DCT had the highest anti-HIV-1 virucidal potency, suggesting that monopeptide 7alpha,12alpha dihydroxy bile salt derivatives have the most potent antiviral activity. Complexing of iodine to the bile salt derivative (as in DICGT) decreases virucidal potency.