Early structural and functional changes in liver of rats treated with a single dose of valproic acid

Valproic acid (VPA) is a simple fatty acid largely used as anticonvulsivant agent. Side effects are uncommon, but cases of fatal hepatic failure have been reported. To elucidate the mechanism of VPA-induced hepatotoxicity, the functional and structural changes associated with administration of sodium valproate (NaVPA) to rats (200 or 600 mg per kg, i.p.) were analyzed. NaVPA produced an immediate, dose-dependent and prolonged increase in bile salt-independent bile flow with a decrease in biliary cholesterol and phospholipid output. At 3 and 5 hr, marked ultrastructural changes were evident in hepatocytes, including formation of autophagic vacuoles engulfing altered mitochondria and occasionally peroxisomes. A modest accumulation of lipoprotein particles was evident at 5 hr in the Golgi cisternae. Twelve-hour samples appeared normal. Bile canaliculi and junctional complexes remained unaltered throughout. The changes observed differ from those previously reported with other hydrocholeretics, such as diethylmaleate; they are likely related to hepatic biotransformation of VPA, which undergoes beta and omega-oxidation, and glucuronidation. While VPA-induced choleresis reflects the physiological osmotic effect of the glucuronide excreted in bile, the ultrastructural changes likely reflect interference by VPA with beta-oxidation of endogenous fatty acids and temporary accumulation of transformation products in the mitochondrial matrix.     

Choleretic effect of valproic acid in the rat

Valproic acid (VPA) is an anticonvulsant agent which produced marked choleresis in the rat. Bile flow rate increased from 50 to 60 microliter per min per kg to 120 to 145 microliter per min per kg immediately after i.v. injection of VPA (37.5 to 150 mg per kg; 2 ml per kg) in male Sprague-Dawley rats. The duration of maximal bile flow was dose-dependent and increased from 30 min (37.5 mg VPA per kg) to approximately 2 hr (150 mg VPA per kg). Choleresis diluted the biliary concentrations of bile acids, Cl-, cholesterol, and phospholipids. VPA did not change the bile/plasma ratio for erythritol suggesting that the increased bile flow is of canalicular origin. VPA did not influence the excretion of bile acids or their osmotic activity, whereas bile salt-independent flow doubled in rats treated with 150 mg VPA per kg. The bile/plasma, bile/liver, and liver/plasma concentration ratios for VPA were 11.7, 1.6, and 7.3, respectively. Approximately 90% of VPA appearing in bile was biotransformed, primarily as a glucuronide. Bile flow correlated with VPA excretion; 16 microliter of bile was produced per micromole VPA which suggests that choleresis is primarily due to the osmotic activity of VPA metabolites in bile. VPA enhanced the excretion of inorganic ions which may also contribute to choleresis. Biliary excretion of phenol-3,6-dibromophthalein disulfonate and ouabain was unaffected. Thus, VPA is an effective choleretic which stimulates bile salt-independent flow of canalicular origin largely as a consequence of the osmotic properties of VPA conjugates in bile.     

Tubulovesicular transcytotic pathway in isolated rat hepatocyte couplets in culture. Effect of colchicine and taurocholate

Isolated rat hepatocyte couplets in short-term culture (6 h) were labeled for 3 min with horseradish peroxidase (HRP) to characterize the transcytotic vesicle transport pathway in this cell culture system that retained an "apical" canalicular membrane polarity. Microtubules were identified with monoclonal antibodies to beta-tubulin and fluorescein iso-thiocyanate-labeled goat-antimouse antibody and were concentrated in the apical domain, a structural polarity that was eliminated by pretreatment with colchicine. In control cells, HRP immediately labeled vesicles and tubules in the submembrane regions of the periphery of the cell. Within 10 min tubules and vesicles were prominently labeled in pericanalicular regions, a process blocked by colchicine but not by lumicolchine or taurocholate administration. A quantitative morphometric analysis utilizing a Zeiss Videoplan-2 image analyzer established that (a) HRP-containing structures increased in density, area, length, and diameter in the pericanalicular region by 10 min; (b) colchicine, but not lumicolchicine, pretreatment diminished their density, area, and length; and (c) taurocholate (50 microM), a choleretic and biliary lipid-stimulating bile acid, had no effect on HRP density or percentage of area in the pericanalicular region, but decreased the diameter of the pericanalicular HRP-containing structures and increased the percentage of tubules containing HRP from 29% to 40%. Tubules were particularly prominent in thick sections (400 nm) in both peripheral and pericanalicular regions and were viewed as continuous anastomosing linear arrays in stereo-paired micrographs. These studies established that isolated rat hepatocyte couplets maintain a highly polarized tubulovesicular transcytotic pathway in short-term culture that is micro-tubule-dependent. Taurocholate stimulates the transformation of tubules from vesicles in this isolated rat hepatocyte couplet system.     

Study on the lipid composition of rat bile during choleresis induced by diethyl maleate

Diethyl maleate (DEM) is known to produce a rapid depletion of hepatic glutathione (GSH) and to induce an immediate short-term choleresis in experimental animals. The aim of our investigation was to ascertain in rats the effect of DEM on biliary lipid composition during the increment in bile flow. Biochemical and morphological studies of liver tissue were conducted in parallel. In bile fistula rats, a doubling of bile flow occurred immediately after intraperitoneal injection of DEM (0.7 ml/kg body weight) returning to the basal level at 45 min. Choleresis diluted the total bile acid concentration, without modifying the secretion. The phospholipid concentration was not affected by DEM, resulting in increased output (p less than 0.02 vs. controls). Both cholesterol concentration and output were significantly decreased during DEM-induced choleresis. These changes were promptly reversible together with the return of bile flow to control values. Biochemical determinations failed to show a DEM-induced modification of enzymatic activities of the microsomal drug biotransformation pathway. Instead, morphological studies revealed alterations of the Golgi apparatus in hepatocytes with marked distension of the cisternae in coincidence with choleresis. The results demonstrate that the bile acid-independent choleresis induced by DEM is accompanied by an alternation in biliary lipid secretion possibly related to an interaction of the drug with the cellular or subcellular membranes of hepatocytes.     

Demonstration of vesicular-dependent bile flow in the sucrose-loaded rat

The contribution of the hepatocyte vacuolar apparatus to bile fluid formation was assessed by studying the transcellular transport and biliary excretion of the fluid-phase marker sucrose. In rats sucrose-loaded by IP administration of sucrose, electron microscopy showed expansion of the vacuolar apparatus and numerous large lysosomelike structures in hepatocytes. Subcellular distribution studies showed that sucrose was sequestered in lysosomes. Compared with controls, sucrose-loaded rats had a 30% higher (P less than 0.01) bile flow with no change in biliary bile acid or electrolyte concentrations. Administration of colchicine ablated the sucrose-induced choleresis and resulted in parallel changes in biliary secretions of sucrose and lysosomal enzymes. Our data suggest that in the sucrose-loaded rat, the hepatocyte vacuolar apparatus may contribute significantly to bile formation by microtubule-dependent release of fluid into bile by exocytosis.     

Insulin-Induced Choleresis in Relation to Insulin Concentrations in Plasma and Bile in the Cat

In experiments on fasting, chloralose-anesthetized cats the concentrations of insulin and glucagon in arterial or portal plasma and bile were studied during insulin-induced choleresis. The administration of insulin in single doses (0.1–1.0 U · kg^-1) and infusions (0.001–0.01 U · kg^-1 · min^-1) resulted in parallel increases in bile flow and ¹⁴C-erythritol clearances of 17%–96%, whereas the net ductular fluid transport and the biliary excretion rate of bile acids remained unchanged. Insulin therefore stimulated the bile acid-independent fraction of the canalicular bile formation. When corrected for the hepatic transit time of insulin (25 min) the initial phase of insulin choleresis was closely related in time to the plasma (30–5000 μU · ml^-1) and biliary (40–210 μU · ml^-1) concentrations of insulin. Bile flow was stimulated even at plasma insulin concentrations similar to those seen postprandially. It is concluded that insulin-induced choleresis to a great extent can be explained by direct, hepatic actions of the hormone, but a delayed release of glucagon may be of importance for the terminal phase of insulin choleresis. The results also demonstrate that insulin crosses the liver by an intercellular pathway. This mode of transport could be of importance for its action on bile production.     

Enhancement by secretin of the apparently maximal hepatic transport of bilirubin in the rat

The effect of secretin (0.4 C.U. per hr per 100 gm body weight) on bile flow and the apparent maximal hepatic transport of bilirubin (Tm) was investigated in the rat. When secretin was administered during an already established bilirubin-Tm condition, it increased bile flow and bilirubin-Tm by 15 to 20% over a 30- to 50-min period. Enhancement of bilirubin output correlated with augmented flow and was sustained by an increased rate of excretion of monoglucuronides. When secretin was given for 90 min before bilirubin loading, it enhanced biliary bilirubin concentration and output, largely as diglucuronides. Bilirubin-Tm correlated positively with glururonyltransferase activity in liver homogenates. In the isolated perfused rat liver, injection of secretin in the portal cannula failed to produce choleresis. Bilirubin uridine diphosphate-glucuronyltransferase activity was lower than in intact rats and higher after treatment than in controls. The effect of secretin had an early effect on hepatocytic bile flow and a later effect on conjugation. Maximal hepatic bilirubin output was modulated both by flow and conjugation rate; the two mechanism may act independently.     

Valproate-induced hepatic steatogenesis in rats

The administration of high-dose valproic acid (VPA) (750 mg per kg) consistently produced significant microvesicular steatosis in mature Sprague-Dawley rats after 48 hr. Similar changes occurred in animals pretreated with phenobarbital which received a lower dose of VPA (350 mg per kg), but no steatosis was seen in animals treated with the low-dose VPA alone. The steatogenic effect of VPA is most likely mediated by a toxic metabolite. It can also be speculated that phenobarbital, by enhancing the inducing effects of the hepatic mixed-function oxidase system, may lead to increased conversion of VPA to a toxic metabolite. Young and weanling rats appeared to be resistant to the steatogenic effects of VPA. Reproduction of microvesicular steatosis in this experimental; model may permit exploration of factors that enhance or inhibit VPA-induced hepatic injury.     

The effect of bilirubin on biliary lipid secretion: Analysis by horseradish peroxidase associated intrahepatic vesicular transport system

The mechanism of biliary lipid secretion is still controverial and there is no definite information regarding how bilirubin inhibits biliary phospholipid and cholesterol secretions without affecting bile salt secretion. In this study, the effects of biliribin on intrahepatic vesicular transport and biliary lipid secretion were examined using bile-fistula rats. Horseradish peroxidase (HRP) was used as a tracer of intrahepatic vesicular transport. Bilirubin (5mg/100g BW) and/or HRP (5mg/100gBW) were injected through the mesenteric vein. Bile flow, biliary bile acid, biliary phospholipid and cholesterol outputs were examined in saline, HRP and HRP+bilirubin groups, respectively. Bile flow and biliary bile acid output were not affected by bilirubin administration. Biliary phospholipid and cholesterol as well as biliary HRP outputs were inhibited just after bilirubin administration, 42.8±6.1 SD% 47.7±5.1 SD%, and 33.4±3.8 SD%, respectively. These results suggested the participation of intrahepatic vesicular transport system in the inhibition of biliary lipid secretion by bilirubin and in its secretory mechanism. This study was partially funded by Uehara Life Science Foundation.     

Sulindac is excreted into bile by a canalicular bile salt pump and undergoes a cholehepatic circulation in rats.

BACKGROUND & AIMS: Dihydroxy bile acids induce a bicarbonate-rich hypercholeresis when secreted into canalicular bile in unconjugated form; the mechanism is cholehepatic shunting. The aim of this study was to identify a xenobiotic that induces hypercholeresis by a similar mechanism. METHODS: Five organic acids (sulindac, ibuprofen, ketoprofen, diclofenac, and norfloxacin) were infused into rats with biliary fistulas. Biliary recovery, bile flow, and biliary bicarbonate were analyzed. Sulindac transport was further characterized using Tr(-) rats (deficient in mrp2, a canalicular transporter for organic anions), the isolated perfused rat liver, and hepatocyte membrane fractions. RESULTS: In biliary fistula rats, sulindac was recovered in bile in unconjugated form and induced hypercholeresis of canalicular origin. Other compounds underwent glucuronidation and were not hypercholeretic. In the isolated liver, sulindac had delayed biliary recovery and induced prolonged choleresis, consistent with a cholehepatic circulation. Sulindac was secreted normally in Tr(-) rats, indicating that its canalicular transport did not require mrp2. In the perfused liver, sulindac inhibited cholyltaurine uptake, and when coinfused with cholyltaurine, induced acute cholestasis. With both basolateral and canalicular membrane fractions, sulindac inhibited cholyltaurine transport competitively. CONCLUSIONS: Sulindac is secreted into bile in unconjugated form by a canalicular bile acid transporter and is absorbed by cholangiocytes, inducing hypercholeresis. At high flux rates, sulindac competitively inhibits canalicular bile salt transport; such inhibition may contribute to the propensity of sulindac to induce cholestasis in patients.     

CCK-receptor antagonists proglumide and loxiglumide stimulate bile flow and biliary glutathione excretion

The mechanism for the choleresis induced by CCK-receptor antagonists, proglumide, loxiglumide, and CR 1409, was examined in anesthetized rats and compared to the effects of CCK itself. These agents were infused intravenously over a 2-hr period, and bile flow, and biliary excretion of bicarbonate, total bile acids, and glutathione were measured in 30-min intervals. All three antagonists produced a dose-dependent choleresis, but a significant decrease in bile acid excretion, indicating that they stimulate bile flow via a bile acid-independent mechanism. The increase in bile flow was associated with a parallel increase in biliary glutathione and biocarbonate output in rats treated with proglumide and loxiglumide. In animals pretreated with acivicin to inhibit gamma-glutamyltransferase activity, proglumide was shown to stimulate biliary excretion of reduced glutathione (GSH), but not glutathione disulfide (GSSG), indicating the absence of oxidative stress in the liver. GSH output was increased by only 0.5–0.9 µmol/30 min after infusion of proglumide at a dose of 75 mg/kg/hr, whereas bile volume was increased 0.2–0.4 ml/30 min, indicating that this increased biliary GSH excretion can account for only a small fraction of the increased bile volume, given an osmotic efficiency for GSH of 34 µl/µmol. In contrast to CCK receptor antagonists, CCK itself had no effect on bile flow and outputs of bicarbonate, GSH, and bile acids, suggesting that the effects of the antagonists are not related to their interaction with CCK receptors. These findings demonstrate that proglumide and loxiglumide stimulate a bile acid-independent bile flow that is only partially explained by an increase in GSH excretion.     

Carbon monoxide-mediated alterations in paracellular permeability and vesicular transport in acetaminophen-treated perfused rat liver.

This study aimed to examine whether acetaminophen (AAP), an anti-inflammatory agent producing hepatocellular damages with its overdose, evokes hepatocellular dysfunction through mechanisms involving carbon monoxide (CO) generated by heme oxygenase (HO). In perfused rat livers, CO and bilirubin were determined in venous perfusate and bile samples as indices of heme degradation. Biliary excretion of transportally injected horseradish peroxidase was also determined to assess paracellular junctional permeability and vesicular transport across hepatocytes. AAP at 20 mmol/L induced a transient choleresis, followed by a reduction of bile output. Under these circumstances, the release of CO and bilirubin IXalpha, terminal products of the HO-mediated heme degradation, became 2. 5-fold greater than the control. The rate of CO production appeared stoichiometric to the degradation rate of microsomal cytochrome P-450. Mechanisms for the AAP-induced cholestasis involved an increase in the junctional permeability that coincided with a reduction of vesicular transport across hepatocytes. Clotrimazole, a cytochrome P-450 inhibitor, or zinc protoporphyrin IX, an HO inhibitor, but not copper protoporphyrin IX, which did not inhibit HO, attenuated these AAP-induced changes. Furthermore, administration of CO at concentrations comparable with those induced by AAP elicited a marked elevation of the paracellular junctional permeability concurrent with a reduction of transcellular vesicular transport, mimicking effects of the AAP administration. Thus, CO serves as a putative regulator of hepatocellular function that is overproduced through acute heme degradation during xenobiotic transformation.     

Stimulation of choleresis by insulin-like growth factor-I in rats

Insulin-like growth factor-I (IGF-I) is an endogenous growth factor which is mainly produced in the liver. The functions of IGF-I can be summarized as growth-promoting and insulin-like metabolic actions. In the present study, the effect of IGF-I on bile flow and bile acid secretion was investigated in rats. In normal rats bile flow was significantly increased by single exogenous administration of IGF-I, and by 1 week treatment of IGF-I, both bile flow and bile acid secretion were significantly increased. Moreover, to further understand the relationship between IGF-I and bile acid secretion, hypophysectomized rats were next used. We found that the decreases in bile flow and bile acid secretion observed in rats after hypophysectomy, as well as the decrease in the endogenous level of IGF-I in the blood, were partially reversed by 1 week exogenous IGF-I treatment. Overall, this study showed that IGF-I stimulates choleresis associated with an elevation of bile acid secretion in both normal and hypophysectomized rats when exogenously administered, suggesting the importance of IGF-I in the stimulation of choleresis in vivo.     

The effect of bilirubin on biliary lipid secretion: analysis by horseradish peroxidase associated intrahepatic vesicular transport system

The mechanism of biliary lipid secretion is still controversial and there is no definite information regarding how bilirubin inhibits biliary phospholipid and cholesterol secretions without affecting bile salt secretion. In this study, the effects of bilirubin on intrahepatic vesicular transport and biliary lipid secretion were examined using bile-fistula rats. Horseradish peroxidase (HRP) was used as a tracer of intrahepatic vesicular transport. Bilirubin (5 mg/100 g BW) and/or HRP (5 mg/100 gBW) were injected through the mesenteric vein. Bile flow, biliary bile acid, biliary phospholipid and cholesterol outputs were examined in saline, HRP and HRP + bilirubin groups, respectively. Bile flow and biliary bile acid output were not affected by bilirubin administration. Biliary phospholipid and cholesterol as well as biliary HRP outputs were inhibited just after bilirubin administration, 42.8 +/- 6.1 SD% 47.7 +/- 5.1 SD%, and 33.4 +/- 3.8 SD%, respectively. These results suggested the participation of intrahepatic vesicular transport system in the inhibition of biliary lipid secretion by bilirubin and in its secretory mechanism.     

The effect of secretin on bile flow and bile acid and bilirubin excretion following relief of prolonged bile duct obstruction in the rat

Bile flow was re-established in rats whose bile ducts had been obstructed for 5, 10, 15 and 28 days (Groups I, II, III and IV, n = 5). The effect of i.v. secretin on bile flow in control rats, whose bile ducts had been cannulated, was minimal, but in cholestatic rats there was an immediate response which was related to the duration of the obstruction and the degree of bile duct proliferation. In 40 min the mean excess bile flow production amounted to 76, 258, 320 and 432 microliters/100 g body wt. in Groups I, II, III and IV, respectively. Choleresis was prolonged in the Group IV rats that had developed cirrhosis. Synthetic secretin had a minimal effect on bile acid and bilirubin excretion. It is postulated that the proliferating bile ductules are the site of secretin choleresis, although the possibility that reduced inactivation of the hormone plays a role cannot be excluded.     

Effects of phalloidin and colchicine on diethylmaleate-induced choleresis and ultrastructural appearance of rat hepatocytes

ABSTRACT: Diethylmaleate is used as a model compound whose glutathione conjugates are secreted into bile, and which induce choleresis and the formation of Golgi-derived vesicles in hepatocytes. This study was performed to test the assumption that these vesicles are involved in the bile canalicular secretion of diethylmaleate. We reasoned that phalloidin and colchicine, two drugs acting on microfilaments and microtubules, respectively, can modify the movements of diethylmaleate-induced vesicles towards the bile canaliculus. Phalloidin induced the formation of a thick microfilamentous network around the bile canalicular plasma membrane domain. A significant decrease in diethylmaleate-stimulated choleresis was observed, associated with a striking accumulation of pericanalicular vesicles, which were confirmed by morphometric analysis. In contrast, in rats pretreated with colchicine, after diethylmaleate administration, only a few vesicles were observed around the bile canaliculus, while diethylmaleate-induced choleresis also decreased. These results suggest that: a) the thick microfilament network induced by phalloidin prevents diethylmaleate-associated vesicles reaching the bile canalicular plasma membrane; and b) colchicine produces a dispersion of these vesicles in the cytoplasm of hepatocytes by inhibiting the polymerization of microtubules. These observations support a role of vesicles in the transport of diethylmaleate by hepatocyte into bile, and are consistent with the existence of a vesicular pathway for the biliary secretion of diethylmaleate and possibly other organic anions.     

Risk Factors of Hyperammonemia in Patients With Epilepsy Under Valproic Acid Therapy

Hyperammonemia has been reported to be associated with patients who receive valproic acid (VPA) therapy. This study aimed to determine the risk factors for hyperammonemia in patients with epilepsy treated with VPA. One hundred and fifty-eight adult patients with epilepsy aged older than 17 years who received VPA therapy were enrolled into this study. Blood samples were taken during the interictal state and analyzed for the blood level of ammonia. Statistical analysis was conducted between different groups of patients. The results showed that the frequency of hyperammonemia associated with VPA therapy was 27.8% (ammonia level >93 µg/dL), and 5.1% of the patients had severe hyperammonemia (ammonia level >150 µg/dL). The blood ammonia level was significantly correlated with the dosage of VPA and the plasma concentration of VPA. An increase of 1 mg in the dosage of VPA increased the risk of hyperammonemia by 0.1%. In addition, combination treatment with liver enzyme inducing antiepileptic drugs (AEDs) and antipsychotic drugs increased the risk of hyperammonemia. In conclusion, the use of VPA in adult patients with epilepsy was associated with a dose-dependent increase in blood concentrations of ammonia. Combination treatment with liver enzyme-inducing AEDs and antipsychotic drugs increased the risk of VPA-induced hyperammonemia. Most of the patients with VPA-induced hyperammonemia were asymptomatic; however, if patients taking VPA present with symptoms such as nausea, fatigue, somnolence, ataxia, and consciousness disturbance, the blood ammonia level should be measured.     

Effect of side-chain shortening on the physiologic properties of bile acids: hepatic transport and effect on biliary secretion of 23-nor-ursodeoxycholate in rodents

To define whether side-chain length influences the physiologic properties of bile acids, nor-ursodeoxycholate (nor-UDC), the C23-nor derivative of ursodeoxycholate (UDC), was synthesized in both nonradioactive and radioactive forms (23-14C). Its hepatic translocation, hepatic biotransformation, and effect on bile flow, biliary bicarbonate, and biliary lipid secretion were compared with that of UDC and those of their respective glycine and taurine conjugates in anesthetized biliary fistula hamsters, rats, and guinea pigs, as well as the isolated perfused hamster liver. Hepatic uptake and biliary output of nor-UDC was slower than that of UDC or cholyltaurine in the isolated perfused hamster liver. In biliary fistula animals, nor-UDC was secreted only in bile. Biliary recovery of nor-UDC as compared to that of UDC was prolonged in the rat and hamster, although not in the guinea pig. Hepatic biotransformation, assessed by chromatography of bile, showed that conjugation of nor-UDC was inefficient, as unconjugated nor-UDC was present in bile; there was little amidation with glycine or taurine in any species, but sulfates and glucuronides, as well as other metabolites, were formed, with the pattern of biotransformation varying among species. When infused over a dosage range of 0.2-30 mumol/kg X min, nor-UDC induced a striking choleresis of canalicular origin. The bile acid-dependent flow was increased threefold in hamsters, ninefold in rats, and nearly twofold in guinea pigs when compared to that induced by UDC. The choleresis was associated with a linear increase in bicarbonate output and concentration in bile, and little phospholipid or cholesterol secretion was induced. A competition experiment in the bile fistula hamster indicated that nor-UDC or its metabolites, or both, appeared to compete for canalicular transport of ursocholyltaurine (a cholyltaurine epimer) when the latter was secreted under its Vmax conditions. Conjugates of nor-UDC and UDC were promptly and almost completely recovered in bile without appreciable hepatic biotransformation; the conjugates did not induce a hypercholeresis or increase biliary bicarbonate concentration. It is proposed that a fraction of nor-UDC is secreted into canalicular bile in the unconjugated form and is protonated by a hydrogen ion derived from carbonic acid that was generated by the hydration of luminal CO2 by carbonic anhydrase present in biliary ductular cells. The protonated bile acid is absorbed, thus generating a bicarbonate anion. The bile acid passes through the cholangiocyte, returns to the sinusoids via the periductular capillary plexus, and is resecreted into bile.(ABSTRACT TRUNCATED AT 400 WORDS)     

An increase of choleresis in secondary biliary cirrhosis in rats is caused by bile duct secretion

Bile flow may be considerably increased in human cirrhosis. The mechanism of this increase has not been established. Two mechanisms have been proposed: a) increased canalicular filtration because of sinusoidal hypertension, or b) secretion by proliferated bile ductules. To distinguish between these two possibilities, we examined the determinants of bile secretion in rats with secondary biliary cirrhosis after bile duct obstruction. Sham-operated animals served as controls. Four weeks after bile duct ligation, all animals had cirrhosis. Bile flow was significantly higher and bile salt secretion significantly lower in cirrhotic animals than in controls. Biliary bicarbonate concentration was significantly higher in cirrhotic animals than in controls. Bile-to-plasma concentration ratio of erythritol was significantly lower in cirrhotic animals than in controls, suggesting a dilution of erythritol by a secretion distal to bile canaliculi. Bile-to-plasma ratio of sucrose was not significantly different in cirrhotics and controls, suggesting that paracellular permeability was not modified. Secretin, at the dose of 3 clinical units/100 g, induced an increase of approximately 75 percent in bile flow, and 70 percent in biliary bicarbonate concentration in cirrhotics. In conclusion, bile flow was increased in biliary cirrhosis in rats. The dilution of erythritol, the increase in biliary bicarbonate concentration and the increased response to secretin strongly suggest that increased choleresis was due, at least in part, to secretion by bile ductules or ducts. These results confirm that secondary biliary cirrhosis is a good experimental model for the study of alterations of bile secretion in cirrhosis.