Erythritol Clearance Research Articles

Hepatotoxicity of an HIV Protease Inhibitor in Dogs and Rats

Oral administration of the HIV protease inhibitor L-689,502 caused cholestasis and hepatocyte injury in rats and dogs. These changes occurred rapidly, with elevations in serum transaminase observed as early as 6 hr after oral dosing in dogs. The acute phase of this hepatotoxic response was characterized in more detail in rats. Following intravenous administration, bile flow was decreased in a dose-dependent manner with greater than 90% decrease in less than 30 min at a dose of 5 mg/kg. The decrease in bile flow was associated with a decrease in erythritol clearance. The decrease in bile flow was not due to disruption of biliary tight junctions. Sucrose clearance was not increased and biliary bile acid concentrations in treated animals were not different from controls. Unlike control animals, bile flow was not stimulated by infusion of the bile acid tauroursodeoxycholic acid in animals treated with L-689,502. These cholestatic effects may be due, in part, to direct hepatocyte injury. Histological examination of perfusion-fixed livers 30 min after L-689,502 administration revealed periportal changes including hepatocyte vacuolation and occasional single cell necrosis. On a subcellular level, the nucleus and mitochondria were intact in less-severely affected cells. However, extensive vacuolation with multilamellar inclusions was pronounced in these cells. In addition, canalicular ectasia was also observed which was consistent with the cholestatic changes that were seen. In summary, L-689,502 is a potent, rapid acting hepatotoxin in dogs and rats. The mechanism by which this agent induces cholestasis is novel compared to other well-characterized cholestatic agents such as α-naphtylisothiocyanate and ethinyl estradiol.

Stimulation of bile acid independent bile flow with bromo-cyclic guanosine monophosphate

The second messenger, cyclic guanosine monophosphate (cGMP), mediates the actions of nitric oxide, natriuretic peptides, and microbial toxins on cellular contractility and electrolyte movement. Because both hepatocellular contractility and electrolyte secretion participate in bile formation, we investigated the actions of cGMP on this process in intact liver. In rat liver perfused with 8-bromo-cyclic GMP (bcGMP) at 0.5 and 3 micromol/min, bile flow increased by 5% and 31%, respectively. The biliary excretion of the bile acid, taurocholate ([3H]-labeled; 1 micromol/min) and of the organic anion, bromosulfophthalein ([35S]-labeled; tracer dose), was unchanged. The paracellular and transcytotic pathways of biliary excretion, assessed by horseradish peroxidase (HRP), were unaffected. BcGMP was concentratively secreted into bile and the accompanying 30% increase in the biliary clearance of erythritol suggested that the choleresis was primarily osmotic in nature. Unlike cyclic adenosine monophosphate (cAMP), which stimulates bile acid dependent bile flow and transcytosis, bcGMP increased bile acid independent bile flow mainly as a result of its concentrative biliary secretion. (Hepatology 1996 Dec;24(6):1487-91)

Stimulation of bile acid independent bile flow with bromo-cyclic guanosine monophosphate.

The second messenger, cyclic guanosine monophosphate (cGMP), mediates the actions of nitric oxide, natriuretic peptides, and microbial toxins on cellular contractility and electrolyte movement. Because both hepatocellular contractility and electrolyte secretion participate in bile formation, we investigated the actions of cGMP on this process in intact liver. In rat liver perfused with 8-bromo-cyclic GMP (bcGMP) at 0.5 and 3 micromol/min, bile flow increased by 5% and 31%, respectively. The biliary excretion of the bile acid, taurocholate ([3H]-labeled; 1 micromol/min) and of the organic anion, bromosulfophthalein ([35S]-labeled; tracer dose), was unchanged. The paracellular and transcytotic pathways of biliary excretion, assessed by horseradish peroxidase (HRP), were unaffected. BcGMP was concentratively secreted into bile and the accompanying 30% increase in the biliary clearance of erythritol suggested that the choleresis was primarily osmotic in nature. Unlike cyclic adenosine monophosphate (cAMP), which stimulates bile acid dependent bile flow and transcytosis, bcGMP increased bile acid independent bile flow mainly as a result of its concentrative biliary secretion.

Plasma and Urine Kinetics of Erythritol after Oral Ingestion by Healthy Humans

The plasma and urine kinetics of erythritol and the effect of erythritol on plasma glucose and insulin levels were studied in human volunteers administered a single oral dose of 1 g erythritol/kg body wt. The plasma level of erythritol increased during the first 30 to 40 min, reaching a maximum value of approximately 2.2 mg/ml after 90 min. Plasma levels of erythritol then declined gradually to approximately 1.5 to 1.7 mg/ml at the end of the 3-hr sampling period. An average of 30% of the ingested amount of erythritol was excreted unchanged in the urine during the first 3 hr. Total urinary excretion increased to 78% after 24 hr. Renal clearance of erythritol was approximately half that of creatinine, indicating tubular reabsorption of erythritol by the kidney. Mean plasma glucose and insulin levels, measured for up to 3 hr after ingestion, were unaffected by erythritol. The results of this study indicate that erythritol was readily absorbed following oral administration and was excreted unchanged in the urine. Less than 20% of erythritol remained unabsorbed and was available for colonic fermentation and potential production of short-chain fatty acids. Its caloric value was estimated to be ≤0.4 kcal/g.

Nitric oxide stimulates bile flow and glutathione excretion independent of cyclic GMP-induced HCO 3- excretion : [formula omitted] Dept. Medicine and Liver Center, Yale University School of Medicine, New Haven, CT and ∗Dept. Gastroenterology, Edith Wolfson Medical Center, Holon, Israel

Based on studies in the pig, secretin choleresis has been proposed to be initiated by colchicine-inhibitable, exocytic insertion into the basolateral cholangiocyte membrane of intracytoplasmatic vesicles containing a H+ ATPase. Formal prof of this hypothesis in the intact liver of other species, however, is lacking. The effect of the microtubule inhibitor colchicine on the ductular bile formation and HCO−3 secretion induced by secretin was, therefore, explored in a secretin-responsive rat model characterized by marked hyperplasia of bile ductules.While colchicine pretreatment significantly decreased basal bile flow from 142.1±8.8 to 83.4±8.2 μl· min−1·kg−1 (p<0.001) and basal biliary erythritol clearance from 112.7±6.3 to 69.9±7.0 μl·min−1. kg−1 (pš0.05), it did not significantly affect basal biliary [HCO−3], nor basal biliary bile acid output. Moreover, colchicine did not alter the effects of secretin. Thus, the secretin-induced increments in bile flow, biliary [HCO−3] and biliary HCO−3 output averaged 58.2±13.6 μl·min−1·kg−1, 16.6±3.1 mM and 5.3±1.4 μmol·min−1·kg−1 in vehicle-pretreated controls and 78.4±12.0 μl·min−1·kg−1, 16.1±2.7 mM and 5.1±0.5 μmol·min−1·kg−1 in colchicine-pretreated animals (all p values=n.s), respectively. This suggests that, at least in the rat model used, microtubule-dependent mechanisms are involved in basal, bile acid independent canalicular, but not in secretin-induced ductular, bile formation. Inasmuch as microtubule-dependent mechanisms are required for vesicle movement, this argues strongly against an absolute requirement for exocytic vesicle insertion in the ductular choleresis induced by secretin.

Colchicine does not inhibit secretin-induced choleresis in rats exhibiting hyperplasia of bile ductules: evidence against a pivotal role of exocytic vesicle insertion

Based on studies in the pig, secretin choleresis has been proposed to be initiated by colchicine-inhibitable, exocytic insertion into the basolateral cholangiocyte membrane of intracytoplasmatic vesicles containing a H+ ATPase. Formal prof of this hypothesis in the intact liver of other species, however, is lacking. The effect of the microtubule inhibitor colchicine on the ductular bile formation and HCO−3 secretion induced by secretin was, therefore, explored in a secretin-responsive rat model characterized by marked hyperplasia of bile ductules.While colchicine pretreatment significantly decreased basal bile flow from 142.1±8.8 to 83.4±8.2 μl· min−1·kg−1 (p<0.001) and basal biliary erythritol clearance from 112.7±6.3 to 69.9±7.0 μl·min−1. kg−1 (pš0.05), it did not significantly affect basal biliary [HCO−3], nor basal biliary bile acid output. Moreover, colchicine did not alter the effects of secretin. Thus, the secretin-induced increments in bile flow, biliary [HCO−3] and biliary HCO−3 output averaged 58.2±13.6 μl·min−1·kg−1, 16.6±3.1 mM and 5.3±1.4 μmol·min−1·kg−1 in vehicle-pretreated controls and 78.4±12.0 μl·min−1·kg−1, 16.1±2.7 mM and 5.1±0.5 μmol·min−1·kg−1 in colchicine-pretreated animals (all p values=n.s), respectively. This suggests that, at least in the rat model used, microtubule-dependent mechanisms are involved in basal, bile acid independent canalicular, but not in secretin-induced ductular, bile formation. Inasmuch as microtubule-dependent mechanisms are required for vesicle movement, this argues strongly against an absolute requirement for exocytic vesicle insertion in the ductular choleresis induced by secretin.

Hepatobiliary function in senescent male Sprague-Dawley rats

The purpose of these studies was to investigate intrahepatic changes underlying age-related decreases in bile flow by evaluating the effects of aging on bile acid-dependent and -independent flow, canalicular versus ductular flow and hepatic tight junction permeability. The isolated perfused liver was used to assess age-related changes in intrinsic hepatobiliary function without the complications of extrahepatic factors such as circulating hormones or hemodynamics. Livers from young adults (3 to 6 mo old) or senescent (22 to 26 mo old) male Sprague-Dawley rats were isolated and perfused in a nonrecirculating, hemoglobin-free system to assess oxygen uptake, bile acid-dependent and -independent bile flow, bile acid uptake, carbon 14-labeled erythritol clearance as a measure of canalicular flow, tight junction permeability and transcellular transport into bile. Rates of oxygen uptake by livers from senescent rats were significantly lower than those of young adults (75 ± 8 μmol/gm/hr vs. 121 ± 5 μmol/gm/hr). Age-related decreases in total bile flow were observed and were associated with similar reductions in 14C-erythritol clearance suggestive of decreased canalicular bile flow. Bile acid-dependent and -independent flow was decreased by 50% and 60%, respectively, in isolated perfused livers from senescent rats. Hepatocellular uptake of taurocholate and rates of bile acid excretion also were about 50% lower in senescent than in young adult rats. Tight junction permeability and transcellular transport were assessed by monitoring appearance of tritiated inulin and horseradish peroxidase in bile after bolus injections of these compounds through the portal vein. Tritiated inulin appearance in bile was decreased slightly in senescent compared with young rats. No age-related differences in tight junction permeability to horseradish peroxidase were observed. In addition, biliary excretion of horseradish peroxidase in livers from senescent rats was not affected by colchicine, suggesting that microtubule-related transport was minimal or absent in the senescent rats. Taken together, these data indicate that decreased rates of bile flow in senescent compared with young rats are caused by age-related effects on intrinsic hepatobiliary function, specifically decreases in canalicular bile flow associated with decreased bile acid-dependent and -independent bile flow. The decreased canalicular bile flow appears to be a result in part of impairment of energy-dependent transport processes and microtubule-dependent transcellular transport across cells. These findings suggest that changes in intrinsic hepatobiliary function occur in aged populations and are not caused exclusively by extrahepatic effects such as altered blood flow or changes in hormonal status. Further, age-related decreases in bile flow may be caused at least in part by decreased active transport of osmotically active solutes into bile.

Hepatobiliary function in senescent male sprague-dawley rats

The purpose of these studies was to investigate intrahepatic changes underlying age-related decreases in bile flow by evaluating the effects of aging on bile acid-dependent and -independent flow, canalicular versus ductular flow and hepatic tight junction permeability. The isolated perfused liver was used to assess age-related changes in intrinsic hepatobiliary function without the complications of extrahepatic factors such as circulating hormones or hemodynamics. Livers from young adults (3 to 6 mo old) or senescent (22 to 26 mo old) male Sprague-Dawley rats were isolated and perfused in a nonrecirculating, hemoglobin-free system to assess oxygen uptake, bile acid-dependent and -independent bile flow, bile acid uptake, carbon 14-labeled erythritol clearance as a measure of canalicular flow, tight junction permeability and transcellular transport into bile. Rates of oxygen uptake by livers from senescent rats were significantly lower than those of young adults (75 +/- 8 mumol/gm/hr vs. 121 +/- 5 mumol/gm/hr). Age-related decreases in total bile flow were observed and were associated with similar reductions in 14C-erythritol clearance suggestive of decreased canalicular bile flow. Bile acid-dependent and -independent flow was decreased by 50% and 60%, respectively, in isolated perfused livers from senescent rats. Hepatocellular uptake of taurocholate and rates of bile acid excretion also were about 50% lower in senescent than in young adult rats. Tight junction permeability and transcellular transport were assessed by monitoring appearance of tritiated inulin and horseradish peroxidase in bile after bolus injections of these compounds through the portal vein. Tritiated inulin appearance in bile was decreased slightly in senescent compared with young rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal Relationship of Changes in Hepatobiliary Function and Morphology in Rats Following α-Naphthylisothiocyanate (ANIT) Administration

These studies were designed to evaluate ANIT-induced changes in both hepatobiliary function and morphology during the onset, progression, and recovery of ANIT-induced cholestasis. A single oral dose of 150 mg/kg of ANIT or vehicle was administered by gavage to male Sprague-Dawley rats and hepatobiliary structure and function were evaluated 16, 24, 48, 72, and 168 hr later. Increased hepatocellular tight junction permeability, increased serum bile acids, and decreased bile acid excretion were observed 16 hr after ANIT administration. At 24 hr, bile flow was decreased in ANIT-treated rats, an effect accompanied by increased tight junction permeability, decreased bile acid excretion, and decreased erythritol clearance (estimate of canalicular flow). In addition, scattered small loci of hepatocellular necrosis accompanied by an inflammatory cell response were observed in ANIT-treated rats at this time, with no microscopic evidence of bile duct obstruction (BDO). These data suggest that the onset of ANIT-induced cholestasis was associated with hepatocanalicular changes and not BDO. In contrast, at 48 and 72 hr after ANIT treatment, cholestasis was more profound and was accompanied by mild hepatocellular necrosis and widespread BDO. Hepatocyte tight junction permeability in ANIT-treated rats was not different from controls at 72 hr. These data suggest that the pathogenesis of ANIT-induced cholestasis is biphasic; the onset of cholestasis appears to be associated with changes in hepatocanalicular function and increased tight junction permeability whereas the later and more profound phase of cholestasis appears to be related to a combination of BDO and hepatocellular dysfunction. The time course of biochemical and morphologic changes following ANIT treatment further suggests that the pathophysiologic changes during the onset or initiation phase of cholestasis differ from those involved in the later and more profound phase of ANIT-induced cholestasis.

Effect of cyclosporine and steroids on canine bile flow

Cyclosporine and hydrocortisone are the main immunosuppressants used in transplant surgery. The purpose of this study was to determine the effect of intravenous administration of cyclosporine and hydrocortisone on bile flow in dogs. Cyclosporine in doses of 0.5, 1.0 and 1.5 mg.kg-1.hr-1 were administered along with 18 mumol/min intravenous sodium taurocholate to dogs with chronic biliary and gastric fistulas. Bile volume and bile chloride concentration and output were increased by cyclosporine in a dose-related manner, whereas bile salt concentration decreased and bile salt output was unchanged. Hydrocortisone produced small but significant increases in bile flow only at the highest dose of hydrocortisone administered. Subsequently, experiments were performed when sodium taurocholate was administered in progressively increasing doses (9, 18 and 36 mumol/min), with the dose changed every hour. Bile volume, [14C]erythritol clearance in bile and bile salt concentrations were measured with and without cyclosporine and hydrocortisone administration. Cyclosporine increased the bile salt-independent fraction of canalicular bile flow and ductular bile flow. Experiments evaluating the role of the cyclosporine carrier polyoxyethylated castor oil (Cremophor EL) demonstrated that this substance had no independent choleretic activity, whereas cyclosporine dissolved in ethanol and administered without Cremophor EL significantly increased bile flow. The results of this study indicate that cyclosporine stimulates chloride-rich choleresis independent of bile salt secretion.

Effect of cyclosporine and steroids on canine bile flow

Cyclosporine and hydrocortisone are the main immunosuppressants used in transplant surgery. The purpose of this study was to determine the effect of intravenous administration of cyclosporine and hydrocortisone on bile flow in dogs. Cyclosporine in doses of 0.5, 1.0 and 1.5 mg · kg−1 · hr−1 and hydrocortisone in doses of 1.25, 2.5 and 5 mg · kg−1 · hr−1 were administered along with 18 μmol/min intravenous sodium taurocholate to dogs with chronic biliary and gastric fistulas. Bile volume and bile chloride concentration and output were increased by cyclosporine in a dose-related manner, whereas bile salt concentration decreased and bile salt output was unchanged. Hydrocortisone produced small but significant increases in bile flow only at the highest dose of hydrocortisone administered. Subsequently, experiments were performed when sodium taurocholate was administered in progressively increasing doses (9, 18 and 36 μmol/min), with the dose changed every hour. Bile volume, [14C]erythritol clearance in bile and bile salt concentrations were measured with and without cyclosporine and hydrocortisone administration. Cyclosporine increased the bile salt-independent fraction of canalicular bile flow and ductular bile flow. Experiments evaluating the role of the cyclosporine carrier polyoxyethylated castor oil (Cremophor EL) demonstrated that this substance had no independent choleretic activity, whereas cyclosporine dissolved in ethanol and administered without Cremophor EL significantly increased bile flow. The results of this study indicate that cyclosporine stimulates chloride-rich choleresis independent of bile salt secretion.

Effect of secretin on bile formation in rats with cirrhosis of the liver: Structure-function relationship

To investigate whether the hypercholeresis seen in cirrhotic humans and animals is due to ductular proliferation or altered inactivation of secretin, or both, we studied the response of bile flow and biliary erythritol clearance to synthetic porcine secretin in rats rendered cirrhotic by chronic exposure to phenobarbital/carbon tetrachloride (n = 11) and untreated control rats (n = 5). Bile duct mass was determined morphometrically. Furthermore, plasma disappearance of secretin was measured by radioimmunoassay. Basal bile flow did not differ between the two groups. Whereas secretin had no effect in the control group, it stimulated bile flow by 49% ± 33% in the cirrhotic group (p < 0.001). Erythritol bile-to-plasma ratio was lower and biliary bicarbonate concentration higher in the cirrhotic rats, suggesting some ductular contribution to bile flow even in the absence of secretin. Biliary bicarbonate concentration did not increase further during secretin administration, whereas bile salt concentration decreased from 27±6 to 18±4 mM. The elimination half-life of secretin was not affected by cirrhosis, averaging 5 ± 2 min in both groups. Bile duct volume was increased in cirrhotics (2.9% ± 1.4% vs. 0.2% ± 0.1%; p < 0.01) and showed an excellent correlation with the maximal secretin-induced increment of bile flow. Our results suggest that the proliferating ductules contribute to bile flow and that increased secretin responsiveness is not due to altered pharmacokinetics in cirrhotic rat liver.

Effects of somatostatin on hepatic bile formation

Somatostatin is a peptide that has anticholeretic properties in the dog. The purpose of the present work was to investigate if somatostatin is an anticholeretic agent in humans also. The effects of intravenous infusion of somatostatin on hepatic bile flow and biliary electrolytes and secretion of biliary lipids were studied in 7 patients with complete biliary drainage who had been operated on for choledocholithiasis. Somatostatin, 250 μg/h, was found to decrease the hepatic bile secretion by ~30%. The peptide also reduced the outputs of bile acids, cholesterol, and phospholipids and the outputs of sodium, potassium, and chloride. The concentrations of the biliary lipids were not significantly changed. Somatostatin inhibited the erythritol clearance in the 2 patients studied by ~25%. The present study thus provides evidence that somatostatin inhibits bile formation in humans. It appears as if the reduction in bile production is mainly due to decreased canalicular bile flow. It is possible that this effect of somatostatin is attributable to inhibition of bile acid synthesis or of transport-secretion of bile acids, or both.

Distal small bowel resection increases mucosal permeability in the large intestine.

Intestinal 14C-erythritol clearance, as a measure of mucosal permeability, was evaluated in rats 4 weeks after distal small bowel resection. In the cecum and colon of resected animals, erythritol clearance was increased both per organ and per unit tissue mass, regardless of the extent of the resection. In the remnant jejunum, however, a small but significant increase in erythritol clearance was only observed after 80% distal small bowel resection, no matter which reference was chosen. This is the first demonstration that intestinal resection increases the mucosal permeability of the remnant small and large intestine.

Canalicular bile flow and bile salt secretion are maintained in rats with liver cirrhosis: Further evidence for the intact cell hypothesis

Different aspects of biliary physiology were studied in rat model of liver cirrhosis induced by CCl4/phenobarbitone. We measured bile flow, bile salt secretion, biliary secretion pressure and bile-to-plasma ratios of inert solutes under basal conditions and during infusion of taurocholate (0.4 and 0.8 mumol.min-1.100 g body wt.-1) in 11 cirrhotic and 10 control male Sprague-Dawley rats. Bile flow and biliary bile salt secretion did not differ between the two groups. Analyzing the relationship between bile salt secretion and bile flow, however, we found an increased slope (P less than 0.02) in the cirrhotic animals, suggesting a higher apparent osmotic activity of the bile salts secreted. Maximal biliary secretion pressure was maintained in cirrhotic animals (22.5 +/- 2.5 vs. 25.0 +/- 2.9 cmH2O) in the absence of exogenous bile salt. During taurocholate infusion it decreased to a lesser extent (P less than 0.001) in cirrhotic animals (13.5 +/- 3.4 vs. 19.3 +/- 3.8 cmH2O). Bile-to-plasma ratios of [3H]sucrose and [14C]ferrocyanide were markedly increased in cirrhotic rats. Biliary [14C]erythritol clearance was equal to bile flow in both groups. In the cirrhotic group, the [3H]sucrose bile/plasma ratio was positively correlated with spleen weight (r = 0.744, P less than 0.01), serum concentration of alkaline phosphatase (r = 0.583, P less than 0.05) and basal maximum biliary secretion pressure (r = 0.801, P less than 0.001). We conclude that chronic portal hypertension is associated with increased permeability of the blood/bile barrier. Nevertheless, bile flow and bile salt secretion are maintained in cirrhotic rats, giving support to the intact cell hypothesis for this important hepatocellular function.

Postnatal development of hepatic bile formation in the rabbit.

To investigate postnatal maturation of hepatic bile formation, bile output was measured in four groups of rabbits: suckling infants at ages 10-14, 18-22, and 26-30 days, and adults. Bile output was collected directly from the common duct during three 1-hr periods: a basal period followed by intravenous infusion of 1 and then 2 mumol/min/kg of glycodeoxycholic acid. [14C]Erythritol and [3H]inulin clearances measured canalicular bile flow and biliary permeability. Under basal conditions and with the exogenous bile acid, bile flow and bile salt secretion were lowest in 10- to 14-day-old infants and showed a gradual increase with increasing age. Bile salt-dependent flow, the linear increase in flow relative to bile salt secretion, was higher in the 10- to 14- and 17- to 22-day-old compared to the adult and 25- to 30-day-old groups. The ratio of chloride to bile salt secreted was also higher in the two younger groups. Bile salt-independent flow at theoretical zero bile salt secretion was absent in the younger groups, but evident in the adult and 25- to 30-day-old rabbits. Canalicular flow estimated by erythritol clearance was linearly related to bile salt secretion. Inulin clearance relative to erythritol clearance was higher in the 10- to 14-day-old infants than the adults. Thus, bile flow and bile salt secretion are reduced in the young infant but rise to near adult levels at the time of weaning, 25-30 days in the rabbit. The increase in flow results from increased bile salt secretion and the appearance of bile salt-independent flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Biliary secretion and hepatic metabolism of taurine-conjugated 7α-hydroxy and 7β-hydroxy bile acids in the dog: Defective hepatic transport and bile hyposecretion

Experiments were carried out using chronic bile fistula dogs to define the physiologic properties and metabolism of two unnatural epimeric monohydroxy conjugated bile acids, 7α-hydroxy cholanoyltaurine and 7β-hydroxy cholanoyltaurine. The compounds, labeled with 14C, were infused intravenously at a rate of 1 μmol/kg · min; effects on bile flow and biliary lipid secretion as well as hepatic biotransformation were defined. The 7-monohydroxy bile acids were secreted quite slowly in bile: recovery during the 90-min infusion interval averaged 16% for the 7α compound and 23% for the 7β compound and after 6 h was only about 60% for the 7α compound and 80% for the 7β compound. Uptake by tissues, presumably the liver, appeared to be efficient, as the level of radioactivity in peripheral blood remained quite low. Both bile acids failed to induce the anticipated increase in bile flow; canalicular bile flow, which was assessed using erythritol clearance, was about half the value observed when cholyltaurine was infused at a similar rate. The “hyposecretion” of bile, which was thought likely to be caused by impaired canalicular transport of the monohydroxy conjugates, was fully reversible, as a subsequent cholyltaurine infusion at a rate of 1 μmol/min · kg immediately restored bile flow and the infused cholyltaurine was secreted normally Each compound was partly 3-hydroxylated during hepatic passage: the 7α compound, about 36% (to form chenodeoxycholyltaurine); the 7β compound, about 23% (to form ursodeoxycholyltaurine). No other biotransformation occurred. Each compound induced phospholipid and cholesterol secretion, but compared to the effects of cholyltaurine, the amount of phospholipid secretion induced (per micromole of secreted bile acid) was less, and that of cholesterol, greater. Thus, the two 7-monohydroxy taurine-conjugated bile acids caused a striking dissociation of induced phospholipid and cholesterol secretion. The results indicate that taurine-conjugated 7-monohydroxy bile acids are poorly secreted by the liver and that their impaired transport is associated with bile hyposecretion, possibly reflecting decreased bile acid-dependent flow; the configuration of their 7-hydroxy group influences their rate of secretion into bile. The results also establish a novel type of bile acid biotransformation (3-hydroxylation) in the dog.

Physiologic determinants of biliary calcium secretion in the dog

Studies were carried out to define the origins and physiologic determinants of biliary calcium secretion in the dog. Canalicular bile flow was varied by infusions of several bile acids or a canalicular choleretic agent, SC2644. Three natural bile acids (cholyltaurine, chenodeoxycholyltaurine, and ursodeoxycholyltaurine) that have a low critical micellar concentration and a synthetic hydroxy-oxo bile acid (12-dehydrocholyltaurine) with a much higher critical micellar concentration were used. Calcium output, bile acid output, and bile flow were determined; canalicular bile flow was assessed by measurement of erythritol clearance. Calcium output increased linearly with bile acid output for all three natural bile acids (r = 0.94; 0.026–0.035 μmolΔcalcium /μmolΔbile acid), but chenodeoxycholyltaurine and ursodeoxycholyltaurine infusions induced a slightly greater secretion of calcium for a given bile acid output and bile flow than cholyltaurine. Calcium output also increased linearly with bile flow, whether bile flow was induced by the canalicular choleretic SC2644 (r = 0.93; 0.0018 μmolΔcalcium/ μlΔwater) or the hydroxy-oxo bile acid (r = 0.99; 0.0021 μolΔcalciumμlΔwater). Because the calcium output induced by the three micelle-forming bile acids was greater than that which could be explained by the induced increase in bile flow per se, it was deduced that canalicular calcium has two origins; a micellar component that is dependent on the presence of micelles or micelle-forming bile acids and an osmotic component that is dependent on osmotic forces, e.g., caused by secretion induced by SC2644 or bile acid molecules and possibly bile acid micelles. A small bile acid-independent output of calcium was also detected and should contribute to the osmotic fraction. To determine whether ductular modification of bile flow influenced biliary calcium secretion, ductular secretion was induced by secretin and ductular absorption was induced by somatostatin. Changes in calcium output followed water movements, increasing with ductular secretion and decreasing with ductular absorption. The relationship between changes in ductular bile flow and calcium output was identical during induced ductular secretion (r = 0.83; 0.0016 μmolΔcalcium/ μlΔwater) or induced ductular absorption (r = 0.91; 0.0019 μmolΔcalcium/μlΔwater). Thus calcium movements in relation to water movements were identical for the osmotic component of canalicular bile and the ductular component of bile. The experimental manipulations in bile secretion caused anticipated changes in biliary calcium concentration which ranged from 1.6 to 6.4 mM. These results indicate that at the usual rates of bile acid secretion, most biliary calcium originates at the canaliculus and is bile acid-dependent. The biliary ductule is permeable to calcium, but the effect of ductular modification of bile flow on biliary calcium outputs is small.

The effects of prostacyclin on canine hepatic bile flow.

The effect of prostacyclin on canine hepatic bile flow was evaluated. Utilizing chronic, unanesthetized bile fistula dogs, prostacyclin was found to significantly increase bile flow rates and bile bicarbonate and cyclic AMP concentration and output when compared to control values. The choleretic response was not due to the effect of the principal metabolic endproduct of prostacyclin, 6-keto-PGF1 alpha, as this substance increased bile flow but did not increase bicarbonate or cyclic AMP concentrations in bile. Prostacyclin did not increase [14C]erythritol clearance in bile suggesting that it stimulates bile duct secretion rather than the bile salt-independent fraction of canalicular bile. To ascertain if a relationship exists between the bicarbonate-rich choleresis produced by prostacyclin and that produced by secretin, the effects of prostacyclin and indomethacin on the choleresis produced by endogenous secretin stimulation were evaluated. During intraduodenal acid infusion, prostacyclin significantly increased bile flow. The increase was not secondary to increased secretin release as plasma secretin levels were not significantly changed from control levels. Prostaglandin synthetase inhibition by indomethacin administration did not alter the choleresis produced by intraduodenal acid. The prostacyclin-induced changes in bile flow were further evaluated in acute experiments in anesthetized dogs during which the effects of prostacyclin on systemic blood pressure, hepatic blood flow and cyclic AMP metabolism could be simultaneously determined. The choleresis produced by prostacyclin occurred in the presence of decreased systemic arterial blood pressure and hepatic blood flow. The increased bile cyclic AMP secretion produced by prostacyclin was not associated with increases in systemic or hepatogenous cyclic AMP. Prostacyclin increases canine bile flow and bicarbonate secretion, a process which may be mediated by cyclic AMP. The choleresis is not secondary to changes in hepatic blood flow or secretin release.

Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver.

The influence of the intracellular glutathione status on bile acid excretion was studied in the perfused rat liver. Perturbation of the thiol redox state by short term additions of diamide (100 microM) or hydrogen peroxide (250 microM) or t-butyl hydroperoxide (250 microM) led to a reversible inhibition of biliary taurocholate release without affecting hepatic uptake; inhibition amounted to 45% for diamide and 90% for the hydroperoxides. Concomitantly, the bile acid accumulated intracellularly. Bile flow increased from 1.3 to 2.0 microliters X min-1 X g liver-1 upon infusion of taurocholate (10 microM); the latter value was suppressed to 1.2 microliters X min-1 X g liver-1 by the addition of t-butyl hydroperoxide (250 microM). Similarly, the hepatic disposition of another bile constituent, bilirubin, was suppressed by 70% upon addition of hydrogen peroxide. While the addition of hydrogen peroxide inhibited also the endogenous release of bile acids almost completely, endogenous bile flow was much less affected, decreasing from 1.3 to 1.0 microliters X min-1 X g liver-1. Measurement of [14C]erythritol clearance showed bile/perfusate ratios of about unity both in the absence and presence of hydrogen peroxide, suggesting canalicular origin of the bile under both conditions. In livers from Se-deficient rats low in Se-GSH peroxidase (less than 5% of controls), hydrogen peroxide inhibited taurocholate transport substantially less, providing evidence for the involvement of glutathione in mediating the inhibition observed in normal livers. The percentage inhibition of taurocholate release and intracellular glutathione disulfide (GSSG) content were closely correlated. The addition of t-butyl hydroperoxide caused a several-fold increase of biliary GSSG release, whereas biliary GSH release was even decreased. The results establish a role of glutathione in canalicular taurocholate disposition.