Dynamics of the conjugate pattern during the infusion of bile acids into isolated rat liver

The conjugate pattern of biliary [14C]bile acids was investigated in isolated perfused rat livers, which were infused with either [24-14C]cholic acid or [24-14C]chenodeoxycholic acid (40 mumol/h) together with or without taurine or cysteine (80 mumol/h). [14C]Bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. The biliary excretion of [14C]bile acids was greater in the infusion with [14C]cholic acid than in the infusion with [14C]chenodeoxycholic acid. Biliary unconjugated [14C]bile acids amounted to about 50% of the total after the infusion with [14C]cholic acid, while only about 10% with [14C]chenodeoxycholic acid. In the initial period of infusion, biliary conjugated [14C]bile acids consisted mostly of the taurine conjugate, which decreased with time and the glycine conjugate increased complementarily. When taurine was simultaneously infused, the decrease in the taurine conjugate was suppressed to some extent. Cysteine infused in place of taurine had a similar influence but was less effective than taurine. The taurine content of liver after the infusion with either of the [14C]bile acids decreased greatly compared with that before the infusion, even when taurine or cysteine was infused simultaneously. The glycine content also decreased after the infusion, but the decrease in glycine was smaller than that in taurine. The results suggest that the conjugate pattern of biliary bile acids in rats depends mainly on the amount of taurine which is supplied to hepatic cells either exogenously from plasma or endogenously within themselves.     

Influence of tauroursodeoxycholic and taurodeoxycholic acids on hepatic metabolism and biliary secretion of phosphatidylcholine in the isolated rat liver

Studies were carried out using an isolated rat liver system to define: the contribution of exogenous phosphatidylcholine (PC) to biliary phospholipid secretion; and its hepatic metabolism during perfusion of the livers with conjugated bile salts with different hydrophilic/hydrophobic properties. A tracer dose of sn-1-palmitoyl-sn-2-[14C]linoleoylPC was injected as a bolus into the recirculating liver perfusate, under constant infusion of 0.75 mumol/min of tauroursodeoxycholate or taurodeoxycholate. The effects on bile flow, biliary lipid secretion, 14C disappearance from the perfusate and its appearance in bile, as well as hepatic and biliary biotransformation were determined. With both the bile salts, about 40% of the [14C]PC was taken up by the liver from the perfusate over 100 min. During the same period less than 2% of the given radioactivity was secreted into bile. More than 95% of the 14C recovered in bile was located within the identical injected PC molecular species. The biliary secretion of labeled as well as unlabeled PC, however, was significantly higher in livers perfused with taurodeoxycholate than tauroursodeoxycholate, while the reverse was observed with respect to bile flow and total bile salt secretion. The exogenous PC underwent extensive hepatic metabolization which appeared to be influenced by the type of bile salt perfusing the liver. After 2 h perfusion, the liver radioactivity was found, in decreasing order, in PC, triacylglycerol, phosphatidylethanolamine and diacylglycerol. In addition, the specific activity of triacylglycerol was significantly higher in tauroursodeoxycholate than in taurodeoxycholate-perfused livers (P less than 0.025), while the reverse was true for the specific activity of hepatic PC (P less than 0.01). Because taurodeoxycholate and tauroursodeoxycholate showed opposite effects on both biliary lipid secretion and hepatic PC biotransformations, we conclude that the hepatic metabolism of glycerolipids is influenced by the physiochemical properties of bile salts.     

Characteristics of sinusoidal uptake and biliary excretion of cysteinyl leukotrienes in perfused rat liver

In single-pass perfused rat liver, the sinusoidal uptake of infused 3H-labelled leukotriene (LT) C4 (10 nmol.l-1) was inhibited by sulfobromophthalein. Inhibition was half-maximal at sulfobromophthalein concentrations of approximately 1.2 mumol.l-1 in the influent perfusate and leukotriene uptake was inhibited by maximally 34%. Sulfobromophthalein (20 mumol.l-1) also decreased the uptake of infused [3H]LTE4 (10 nmol.l-1) by 31%. Indocyanine green (10 mumol.l-1) inhibited the sinusoidal [3H]LTC4 uptake by 19%. Replacement of sodium in the perfusion medium by choline decreased the uptake of infused [3H]LTC4 (10 nmol.l-1) by 56%, but was without effect on the uptake of sulfobromophthalein. The canalicular excretion of LTC4, LTD4 and N-acetyl-LTE4 was inhibited by sulfobromophthalein. In contrast, the proportion of polar omega-oxidation metabolites recovered in bile following the infusion of [3H]LTC4 was increased. Taurocholate, which had no effect on the sinusoidal leukotriene uptake, increased bile flow and also the biliary elimination of the radioactivity taken up. With increasing taurocholate additions, the amount of LTD4 recovered in bile increased at the expense of LTC4. Following the infusion of [3H]LTD4 (10 nmol.l-1), a major biliary metabolite was LTC4 indicating a reconversion of LTD4 to LTC4. In the presence of taurocholate (40 mumol.l-1), however, this reconversion was completely inhibited. The findings suggest the involvement of different transport systems in the sinusoidal uptake of cysteinyl leukotrienes. LTC4 uptake is not affected by bile acids and has a sodium-dependent and a sodium-independent component, the latter probably being shared with organic dyes. Sulfobromophthalein also interferes with the canalicular transport of LTC4, LTD4 and N-acetyl-LTE4, but not with the excretion of omega-oxidized cysteinyl leukotrienes. The data may be relevant for the understanding of hepatic leukotriene processing in conditions like hyperbilirubinemia or cholestasis.     

Effect of diabetes on the metabolism of chenodeoxycholic acid in isolated perfused rat liver

The formation of alpha-muricholic acid and beta-muricholic acid from chenodeoxycholic acid was comparatively investigated in livers isolated from normal, streptozotocin-diabetic, and insulin-treated diabetic rats. [24-14C]Chenodeoxycholic acid or [24-14C]alpha-muricholic acid was infused into the perfused livers. There was no difference in biliary excretion of 14C among the different groups of rats after the infusion of each 14C-labelled bile acid. Biliary [14C]bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. In the diabetic group, the formation ratio of alpha-muricholic acid and beta-muricholic acid from [24-14C]chenodeoxycholic acid and also that of beta-muricholic acid from [24-14C]alpha-muricholic acid were much smaller than in the normal group. Treatment of the diabetic group with insulin cancelled the difference in the infusion of each [24-14C]bile acid. The results indicate that not only 6 beta-hydroxylation of chenodeoxycholic acid to alpha-muricholic acid but also 7-epimerization of the latter acid to beta-muricholic acid is suppressed in an insulin-deficient state in rats.     

Metabolism of bile alcohols in the perfused rabbit liver.

The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-3H]cholic acid was observed when the liver was perfused with 5beta-[G-3H]cholestane-3alpha, 7alpha-diol, 5beta-[G-3H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol. No [G-3H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates.     

Bile salts in submicellar concentrations promote bidirectional cholesterol transfer (exchange) as a function of their hydrophobicity

Cholesterol, despite its poor solubility in aqueous solutions, exchanges efficiently between membranes. Movement of cholesterol between different subcellular membranes in the hepatocyte is necessary for assembly of lipoproteins, biliary cholesterol secretion, and bile acid synthesis. Factors which initiate and facilitate transfer of cholesterol between different membranes in the hepatocyte are incompletely understood. It is known that cholesterol secretion into the bile is linked to bile salt secretion. In the present study, we investigated the effects of bile salts of different physicochemical properties at submicellar concentrations (150- 600 microM) on the transfer of [14C]cholesterol from hepatocytes, or crude hepatocellular membranes (donors), to rat high density lipoproteins (acceptor). Bile salts included taurine conjugates of ursodeoxycholic acid (TUDCA), hyodeoxycholic acid (THDCA), cholic acid (TCA), chenodeoxycholic acid (TCDCA), and deoxycholic acid (TDCA). High density lipoprotein (HDL) was separated from hepatocellular membranes and the transfer of [14C]cholesterol from the membranes to HDL was quantitatively determined. In the absence of HDL, [14C]cholesterol remained confined to the membrane fraction. Following addition of HDL, [4-14C]cholesterol in the HDL fraction increased linearly over time. Addition of hydrophilic bile salts (TUDCA and THDCA) increased transfer of [4-14C]cholesterol to HDL only minimally. By contrast, more hydrophobic bile salts stimulated transfer of labeled cholesterol to HDL, and their potency increased in order of increasing hydrophobicity (TCA less than TCDCA less than TDCA). Both for single bile salts and mixtures of bile salts at a total bile salt concentration of 0.30 mM, the rate of cholesterol transfer exhibited a strong linear correlation with a bile salt monomeric hydrophobicity index (r = 0.95; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature on the degradation and biliary secretion of asialoorosomucoid by the perfused rat liver: evidence for two intracellular pathways

We have utilized the in situ perfused rat liver under nonrecirculating conditions to examine the effect of temperature on the metabolism and biliary secretion of [125I]-asialoorosomucid (ASOR). In this manner we were able to follow the fate of a single round of internalized ligand. In control livers perfused at 37 degrees C, approximately 50% of [125I]-ASOR injected into the portal vein was extracted on first pass. Five minutes after the injection, radioactivity, which had been extracted initially, began to appear in the hepatic venous effluent. Within 25 min, 50% of the initially extracted radioactivity was released into the perfusion medium; the bulk of this radioactivity (greater than 95%) was soluble in trichloroacetic acid. In livers perfused at temperatures slightly less than 37 degrees C (30-35 degrees C), first-pass extraction of [125I]-ASOR was similar to that observed at 37 degrees C. However, a severalfold decrease in the rate of release of radioactivity from the liver into the perfusion medium was noted at the lower perfusion temperatures; whereas greater than 50% of the initially extracted radioactivity was released within 30 min from livers perfused at 37 degrees C, only 5% was released at 30 degrees C. At the lower perfusion temperature, a larger proportion of the released radioactivity was acid precipitable (24% vs. 5%). Some radioactivity also was recovered in the bile; of the total amount of radioactivity released from the liver in 30 min at 37 degrees C, approximately 5% was directed into the bile. At lower temperatures of perfusion, a greater fraction of the radioactivity that was released from the liver was directed into the bile (20% at 30 degrees C vs. 5% at 37 degrees C). The data imply that the endosomal pathway to the lysosome is highly sensitive to slight reductions in temperature while the transcytotic route into bile is less sensitive. Lower temperatures might prolong the residence time of ASOR in the prelysosomal endosomal compartments, and thereby increase the likelihood that undegraded ligand will be returned to the blood or be missorted into bile.     

Effect of anisotonic cell-volume modulation on glutathione-S-conjugate release, t-butylhydroperoxide metabolism and the pentose-phosphate shunt in perfused rat liver.

1. Addition of 1-chloro-2,4-dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione-S-conjugate [S-(2,4-dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Anisotonic perfusion did not affect total S-conjugate formation, but release of the S-conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S-conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume-regulatory net K+ uptake. 2. Basal steady-state release of oxidized glutathione (GSSG) into bile was 1.30 +/- 0.12 nmol.g-1.min-1 (n = 18) during normotonic (305 mOsmol/l) perfusion and was 3.8 +/- 0.3 nmol.g-1.min-1 in the presence of t-butylhydroperoxide (50 mumol/l). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t-butylhydroperoxide (50 mumol/l)-stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/l) increased. Anisotonic exposure was without effect on t-butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver-cell swelling due to addition of glutamine plus glycine (2 mmol/l, each). 3. Hypotonic exposure led to a persistent stimulation 14CO2 production from [1-14C]glucose by about 80%, whereas 14CO2 production from [6-14C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited 14CO2 production from [1-14C]glucose by about 40%, whereas 14CO2 production from [6-14C]glucose was unaffected. The effect of anisotonicity on 14CO2 production from [1-14C]glucose was also observed in presence of t-butylhydroperoxide (50 mumol/l), which increased 14CO2 production from [1-14C]glucose by about 40%. 4. t-Butylhydroperoxide (50 mumol/l) was without significant effect on volume-regulatory K+ fluxes following exposure to hypotonic (225 mOsmol/l) or hypertonic (385 mOsmol/l) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t-butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions. 5. The data suggest that hypotonic cell swelling stimulates flux through the pentose-phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide-induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione-S-conjugates into the sinusoidal circulation at the expense of biliary secretion.     

Antiarrhythmic drugs impair hepatic uptake and secretory function by different mechanisms in the isolated perfused rat liver

In the present study the effect of various antiarrhythmic drugs on hepatic perfusion parameters, uptake capacity of organic anions and biliary secretion using the isolated perfused rat liver was examined. Infusion of verapamil (VP), diltiazem, N-propyl-ajmaline (NPAB), and quinidine at pharmacological doses induced consistently a 1.4-1.6-fold increase in portal pressure accompanied by a approximately 60% decrease in bile flow and a approximately 65% inhibition of biliary taurocholate (TC) excretion. Furthermore, hepatic uptake of oxygen, bromosulphthalein (BSP), and TC was significantly reduced. All these effects were dose-dependent and reversible upon withdrawal of the drugs. Studies of the hepatic circulation using a Trypan blue staining technique demonstrated a patchy perfusion pattern during infusion of the antiarrhythmic drugs as compared to the homogenously stained control organ. The hemodynamic alterations and the impairment of the hepatic initial uptake function could be entirely prevented by concomitant administration of the vasodilator papaverine. Bile flow and biliary TC excretion, however, were still inhibited under these conditions. The present results indicate that antiarrhythmic drugs produce cholestasis in the isolated perfused rat liver independently of their adverse effect on hepatic hemodynamics.     

The synthesis of bile acids in perfused rat liver subjected to chronic biliary drainage

1. Isolated rat liver was perfused with heparinized whole blood under physiological pressure resulting in the secretion of bile at about the rate observed in vivo. 2. The preparation remained metabolically active for 4h and was apparently normal in function and microscopic appearance. 3. When the perfusate plasma and liver cholesterol pool was labelled by the introduction of [2-(14)C]mevalonic acid the specific radioactivity of the perfusate cholesterol increased. The biliary acids (cholic acid and chenodeoxycholic acid) were labelled and had the same specific radioactivity. 4. Livers removed from rats immediately after, and 40h after, the start of total biliary drainage, were perfused; increased excretion rates of both cholic acid and chenodeoxycholic acid were found when the liver donors had been subjected to biliary drainage. 5. The incorporation of [2-(14)C]mevalonic acid or rat lipoprotein labelled with [(14)C]cholesterol into bile acids was studied. 6. A dissociation between the mass of bile acid excreted and the rate of incorporation of (14)C was found. This was attributed to the changing specific radioactivity of the cholesterol pool acting as the immediate bile acid precursor.     

Metabolism of cysteinyl leukotrienes in non-recirculating rat liver perfusion. Hepatocyte heterogeneity in uptake and biliary excretion

1. The uptake, metabolism and biliary excretion of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, were studied in a non-recirculating rat liver perfusion system at constant flow in both antegrade (from the portal to the caval vein) and retrograde (from the caval to the portal vein) perfusion directions. During a 5-min infusion of [3H]LTC4, [3H]LTD4 and [3H]LTE4 (10 nmol/l each) in antegrade perfusions single-pass extractions of radioactivity from the perfusate were 66%, 81% and 83%, respectively. Corresponding values for LTC4 and LTD4 in retrograde perfusions were 83% and 93%, respectively, indicating a more efficient uptake of cysteinyl leukotrienes in retrograde than in antegrade perfusions. The concentrations of unmetabolized leukotrienes in the effluent perfusate were 8-12% in antegrade and 2-4% in retrograde perfusions. [14C]Taurocholate extraction from the perfusate was inhibited by LTC4 by only 3%, suggesting that an opening of portal-venous/hepatic-venous shunts does not explain the effects of perfusion direction on hepatic LTC4 uptake. 2. Following infusion of [3H]LTC4 and [3H]LTD4, in the antegrade perfusion direction, about 80% and 87%, respectively, of the radiolabel taken up by the liver was excreted into bile. In retrograde perfusions, however, only 40% and 57%, respectively, was excreted into bile and the remainder was slowly redistributed into the perfusate, indicating that leukotrienes were taken up into a hepatic compartment with less effective biliary elimination or converted to metabolites escaping biliary excretion. The metabolite pattern found in bile was not affected by the direction of perfusion. Biliary products of LTC4 were polar metabolites (31-38%), LTD4 (27-30%), LTE4 (about 1%) and N-acetyl-LTE4 (3-4%) in addition to unmodified LTC4 (17-18%). 3. LTC4 was identified as a major metabolite of [3H]LTD4 in bile, amounting to about 20% of the total radioactivity excreted into bile. This is probably due to a gamma-glutamyltransferase-catalyzed glutamyl transfer from glutathione in the biliary compartment, as demonstrated in in vitro experiments. The presence of sinusoidal gamma-glutamyltransferase activity in perfused rat liver was shown in experiments on the hydrolysis of infused gamma-glutamyl-p-nitroanilide. 90% inhibition of this enzyme activity by AT-125 did not affect the metabolism of LTC4. 4. When [3H]LTE4 was infused in the antegrade perfusion direction, biliary metabolites comprised N-acetyl-LTE4 (24%) and polar components (60%).(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of acetone and conversion of acetone to acetate in the perfused rat liver

The utilization of millimolar concentrations of [2-14C]acetone and the production of acetone from acetoacetate were studied in perfused livers from 48-h starved rats. We devised a procedure for determining, in a perfused liver system, the first-order rate constant for the decarboxylation of acetoacetate (0.29 +/- 0.09 h-1, S.E., n = 8). After perfusion of livers with [2-14C]acetone, labeled acetate was isolated from the perfusion medium and characterized as [1-14C]acetate. No radioactivity was found in lactate or 3-hydroxybutyrate. After 90 min of perfusion with [2-14C]acetone, the specific activity of acetate was 30 +/- 4% (n = 13) of the initial specific activity of acetone. We conclude that, in perfused livers from 2-day starved rats, acetone metabolism occurs for the most part via free acetate.     

Estimation of bile acid pool sizes from their spillover into systemic blood

We have examined the possibility of assessing primary bile acid pool sizes from the spillover of the bile acids into systemic blood after intestinal exposure to the total endogenous bile acid pool; the studies were carried out in 16 healthy subjects. Bile acid spillover was calculated as the integrated area under the curve of bile acid conjugates in serum of each primary bile acid class in response to a well-defined sustained cholecystokinin-induced stimulus of the enterohepatic circulation for 55 min causing complete gallbladder emptying. Serum levels of each species of primary bile acid conjugates were measured by two specific and sensitive radioimmunoassays, one for conjugated cholate and one for conjugated chenodeoxycholate. Primary bile acid pool sizes determined with [24-14C]cholic acid and [24-14C]chenodeoxycholic acid according to Lindstedt (1957. Acta Physiol. Scand. 40:1-9) served as reference. Bile acid conjugates of both species reached a peak 70 min after the start of the cholecystokinin infusion, probably reflecting simultaneous intestinal absorption of both primary bile acids in this model. Highly significant linear correlations were found between the integrated areas under the curve and primary bile acid pool sizes, which were closer for chenodeoxycholate (n = 16, r = 0.81, P less than 0.001) than for cholate (n = 16, r = 0.74, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Peroxisomal bile acid-CoA:amino-acid N-acyltransferase in rat liver

Bile acid-CoA:amino-acid N-acyltransferase activity was measured in subcellular fractions of rat liver homogenate. The conversion of [14C]choloyl-CoA and [14C]chenodeoxycholoyl-CoA into the corresponding [14C]tauro- and glyco-bile acids was calculated after isolation of the product by high performance liquid chromatography. There was an enrichment of bile acid-CoA:amino-acid N-acyltransferase activity in the light mitochondrial (L) fraction and to a lesser extent in the microsomal fraction. Surprisingly, no enrichment was found in the cytosolic fraction. Subfractionation of the L-fraction by Nycodenz gradient centrifugation, showed that the activity of the N-acyltransferase had a bimodal distribution and co-sedimented with peroxisomes (particulate catalase) and microsomes (esterase). The highest specific amidation activity of both choloyl-CoA and chenodeoxycholoyl-CoA was always found in the most peroxisome-rich fractions. [14C]Taurocholate formation in the peroxisomal fraction was 2.2 mumol/mg of protein/min. Striking differences were observed in the Km values and the saturation concentrations for glycine and taurine. The peroxisomal amidation of [14C]choloyl-CoA had a Km for taurine of 0.9 x 10(-3) M and for glycine of 17 x 10(-3) M. The results are consistent with the possibility that most of de novo synthesized bile acids conjugate to taurine by a peroxisomal bile acid-taurine N-acyltransferase in rat liver. The bile acids deconjugated in the gut and recirculating to the liver may be activated and amidated by the microsomal enzyme system prior to biliary secretion.     

Bile acids. 38. Conversion of 5 -cholestane-3 ,7 -diol to allo bile acids by the rat

5alpha-[4-(14)C, 3alpha-(3)H]Cholestane-3beta,7alpha-diol was prepared from individual samples of 5alpha-[3alpha-(3)H]cholestane-3beta,7alpha-diol and 5alpha-[4-(14)C]cholestane-3beta,7alpha-diol, each derived from 3beta-acetoxycholest-5-en-7-one. Bile was collected for 11 days from adult male rats, with cannulated bile ducts, that had received intraperitoneally 0.90-0.92 mg of the doubly labeled diol. Bile from the first 10 hr, containing 63% of the administered (14)C and 6% of the (3)H, was hydrolyzed, and the bile acids were separated by acetic acid partition chromatography. Allochenodeoxycholic and allocholic acids contained at least 20.6% and 48.6%, respectively, of the (14)C retained in the biliary acids. Small amounts of (14)C (2.5% and 1.9%, respectively) were present in the 3beta isomers of these acids, but the tritium content totaled more than half of that found in the bile acid fraction. No evidence was obtained for presence of the extensive quantities of the allomuricholates.     

The transport of lipoprotein cholesterol into bile: a reassessment of kinetic studies in the experimental animal

Studies were undertaken to assess the contribution of lipoprotein cholesterol to bile and to determine whether already-existent hepatic free cholesterol and the free cholesterol which is newly generated from the hydrolysis of hepatic cholesteryl esters are equally available for secretion into bile or constitute metabolically separate pools. Rats with a bile fistula were injected with an intravenous bolus of high-density lipoprotein recombinants containing free [14C]cholesterol and [3H]cholesteryl esters. Results showed (1) that bile free [14C]cholesterol secretion was a constant and linear proportion of the whole liver free [14C]cholesterol pool, (2) that secretion into bile of free [3H]cholesterol was in direct proportion to the rate of hydrolysis of hepatic [3H]cholesteryl esters, and (3) that rates of biliary cholesterol secretion were very similar when secretion was calculated using the specific activity of free [14C]cholesterol and free [3H]cholesterol in the entire liver to 'label' the precursor free cholesterol pool. Furthermore, rates of secretion that were calculated using either isotope closely approximated the mass of free cholesterol that was directly measured in bile. Results thus indicate that because of equilibration and extensive dilution by the large pool of already-existent free cholesterol, the transport of isotopic cholesterol from lipoproteins cannot be used to directly assess the contribution of lipoprotein cholesterol to the cholesterol that is secreted in bile. These studies further suggest that the totality of preformed free cholesterol in the liver is in metabolic equilibrium in one single kinetic pool and that all hepatic free cholesterol is potentially available for secretion into bile.     

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.     

Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]testosterone

1. [4-(14)C]Testosterone was administered intravenously to anaesthetized male cats as a single injection or as a 45-60min. infusion. 2. Most of the administered radioactivity was excreted in the bile (70-80%); only 2.9-5.5% of the dose was excreted in the urine. 3. Bile and urine samples were hydrolysed successively to yield glucuronide, ;cold-acid-hydrolysed' and ;hot-acid-hydrolysed' fractions. 4. The proportion of glucuronides in bile decreased in successive samples, but cold-and hot-acid-hydrolysed metabolites showed no consistent change. 5. After hydrolysis most of the radioactivity in both bile and urine could not be extracted by ether from neutral aqueous solution.     

Bile acid structure and bile formation in the guinea pig

The effects of intravenous infusions (1-4 mumol/min/kg) of 14 bile acids, cholic, deoxycholic, ursodeoxycholic, chenodeoxycholic, dehydrocholic, and their glycine and taurine conjugates, on bile flow and composition and on the biliary permeation of inert carbohydrates have been studied in the guinea pig bile fistula. Hydroxy bile acids were eliminated in bile without major transformation, except for conjugation (over 90%) when unconjugated bile acids were infused. During infusion of dehydrocholate and taurodehydrocholate, 77-100% of the administered dose was recovered in bile as 3-hydroxy bile acids, thus indicating that reduction of the keto group in position 3 was virtually complete. All bile acids produced choleresis at the doses employed: the strongest choleretic was deoxycholate (81.78 microliters/mumol), the weakest was taurodehydrocholate (10.2 microliters/mumol). Choleretic activity was directly and linearly related to bile acid hydrophobicity, as inferred by HPLC, both for similarly conjugated bile acids, and for bile acids having the same number, position, or configuration of the hydroxyl groups. In all instances, the rank ordering was: deoxycholate greater than chenodeoxycholate greater than cholate greater than ursodeoxycholate. During choleresis produced by any of the bile acids tested, bicarbonate concentration in bile slightly declined, but the calculated concentration in bile-acid-stimulated bile (45-57 mmol/l) was always higher than that measured in plasma (23-26 mmol/l). Biliary concentrations of cholesterol (20-68 mumol/l) and phospholipid (14-63 mumol/l) were very low during spontaneous secretion, and declined even further following bile acid choleresis. None of the infused bile acids consistently modified biliary excretion of cholesterol and phospholipid. Consistent with a previous observation from this laboratory, all hydroxy bile acids reversibly diminished [14C]erythritol and [14C]mannitol biliary entry during choleresis, while they increased or failed to modify that of [3H]sucrose and [3H]inulin. The rank ordering for the inhibitory effect on [14C]erythritol and [14C]mannitol permeation was: 3 alpha,7 alpha,12 alpha-trihydroxy greater than 3 alpha,7 alpha-dihydroxy greater than 3 alpha,7 beta-dihydroxy greater than 3 alpha,12 alpha-dihydroxy bile acids.(ABSTRACT TRUNCATED AT 400 WORDS)