Gangliosides released by the perfused rat liver are associated to high density lipoprotein.

We examine here the delivery of gangliosides from the perfused rat liver into the perfusate. One hour after the administration of [3H]GM1 to recirculating perfused livers, almost 80% of the perfusate radioactive gangliosides were recovered associated to the HDL fraction. This fraction was relatively enriched in radioactive GD1a. The pattern of endogenous gangliosides from perfused livers, rat serum and perfusates were very different: GM3 was the main liver ganglioside, GM1 and GD1a were the most abundant in perfusates being GM3 almost absent; GM3, GM1 and GD1a were present in rat serum in similar proportions. Using a non-recirculating perfusion protocol, radioactive gangliosides were found in the HDL fraction since 15 minutes after the administration of [3H]GM1. These results suggest that rat liver supplies the perfusates with some gangliosides and that they are associated to HDL. These facts arise the possibility that the liver is one of the source of serum gangliosides.     

Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers

Because of the low hepatic activity of gamma-glutamyl-transferase in the rat, the liver is generally considered to play only a minor role in the degradation of glutathione conjugates, a limiting step in mercapturic acid formation. Recent findings indicate, however, that the liver has a prominent role in glutathione catabolism, particularly in species other than rat. To examine the contributions of liver to mercapturic acid biosynthesis, mercapturate formation was compared in isolated perfused livers from rats and guinea pigs dosed with either 0.3 or 3.0 mumol of 1-chloro-2,4-dinitrobenzene (CDNB). Chemically synthesized glutathione conjugate, mercapturic acid, and intermediary metabolites of CDNB were used as standards in the high performance liquid chromatography analysis of bile and perfusate samples. Biliary excretion accounted for almost all of the recovered metabolites. A marked species difference was observed in the pattern of CDNB metabolism. Rat livers dosed with 0.3 mumol of CDNB excreted 55% of total biliary metabolites as the glutathione conjugate and 8.2% as the mercapturic acid, whereas guinea pig livers excreted only 4.8% as the glutathione conjugate and 47% as the mercapturate. Mercapturic formation was also dose-dependent, with a larger fraction formed at the 0.3- versus the 3.0-mumol dose (8.2 versus 3.7% in the rat; 47 versus 19% in the guinea pig). Hepatic conversion of the glutathione conjugate to the mercapturic acid was markedly inhibited in both species after retrograde intrabiliary infusion of acivicin, an inhibitor of gamma-glutamyltransferase activity. These findings provide direct evidence for intrahepatic biosynthesis of mercapturic acids. Thus, glutathione conjugates synthesized within hepatocytes are secreted into bile and broken down to cysteine conjugates; the latter are then presumably reabsorbed by the liver, N-acetylated to form the mercapturic acid and re-excreted into bile.     

Triamcinolone activation of renal ammonia production.

The effects of scillaren and dinitrophenol on bilirubin excretion by the perfused rat liver were studied. Both compounds inhibited bile flow, scillaren by 20 to 40%, and dinitrophenol by 60 to 80%. Bilirubin excretion was also impaired. However, the effect of scillaren on bilirubin excretion was less than that on bile flow, as indicated by an increase in the bile bilirubin concentration, whereas dinitrophenol had a greater effect on bilirubin excretion than on bile flow. Dinitrophenol also inhibited the hepatic removal of unconjugated bilirubin from the perfusate, probably because it impaired the initial uptake and/or storage of unconjugated bilirubin by the perfused liver.     

Output and effects of thromboxane produced by the liver perfused with phorbol myristate acetate

The capacity of the perfused rat liver to produce thromboxane after stimulation by phorbol myristate acetate was examined. A total of 109 +/- 20 and 155 +/- 28 pmol/g liver were found in the perfusate and in the bile, respectively, after 40 min. The amount of thromboxane recovered in the perfusate and in the bile accounted for 12.6% of the production calculated from the same number of Kupffer cells in primary cultures, indicating that a major part of thromboxane was taken up and inactivated by hepatocytes. The effect of endogenously synthesized thromboxane on the liver was assessed by using CGS 13080, a thromboxane synthase inhibitor, or BM 13.177, a thromboxane receptor antagonist. 20 nM CGS 13080 in the perfusate inhibited the synthesis of thromboxane and at the same time the elevation of portal pressure and glycogenolysis following administration of phorbol 12-myristate 13-acetate (PMA). The thromboxane receptor antagonist BM 13.177 did not inhibit the synthesis of thromboxane, but reduced the PMA-related elevation of portal pressure and glycogenolysis to the same extent (greater than 60%) as CGS 13080. Sodium nitroprusside, a vasodilator, inhibited the rise in portal pressure caused by PMA to the same extent as CGS 13080 or BM 13.177 but reduced the increase in glycogenolysis only by 25%. These results indicate that thromboxane released by stimulated Kupffer cells of the liver elevates portal pressure and glycogenolysis in the perfused rat liver, although by different mechanisms.     

The role of conjugation reactions in enhancing biliary secretion of bile acids.

Shortening the five-carbon carboxylic acid side chain of cholic acid by one methylene group gave rise to a bile acid (norcholate) that was not a substrate for the bile acid-conjugating enzymes. The metabolism and biliary secretion of norcholate in intact liver was examined in the isolated perfused rat liver system. When rat livers were perfused with 14-20 microM solutions of norcholate for 10 min, norcholate was found in the unconjugated form in liver, venous effluent and bile. Neither tauronorcholate nor glyconorcholate was detectable by high-pressure liquid chromatography or fast-atom-bombardment mass spectrometry. The kinetics of hepatic uptake and biliary secretion of norcholate was compared with that for cholate, taurocholate and chemically synthesized tauronorcholate. The latter three bile acids were completely cleared from the perfusate and efficiently secreted into the bile. However, norcholate was incompletely extracted from the perfusate, and this was shown to be at least partially due to its relatively lower rate of hepatic uptake. Furthermore, the rate of norcholate secretion into bile was greatly reduced relative to the secretion of cholate or chemically synthesized tauronorcholate, even though the concentration of norcholate in the liver was comparatively high. These data demonstrate that the conjugation of bile acids greatly facilitates their secretion into bile.     

Prevention of the toxicity of erythromycin estolate in the perfused rat liver by endotoxin

The possibility that endotoxin pretreatment could prevent the hepatotoxic effects of erythromycin estolate (EE) was investigated using the isolated perfused rat liver. The addition of E. coli endotoxin (25 micrograms/ml) to the perfusate, 30 min prior to EE administration at 150 or 200 microM, significantly ameliorated the decreases in bile and perfusate flow caused by either concentrations of the drug in control liver preparations. This phenomenon was also studied using liver isolated from rats pretreated in vivo with endotoxin for three days. In these preparations, EE at both concentrations did not alter bile flow and caused reductions of perfusate flow which were far less than those observed in untreated control livers. Furthermore, in livers from endotoxin-treated rats EE induced less reduction of bile acid excretion and, at 150 microM, it did not increase the bile to perfusate ratio of sucrose seen in control preparations after the drug, which may be an expression of altered hepatocytic membrane permeability. Since it is known that both endotoxin and EE interact with membranes, it is suggested that the "protective" effects of endotoxin may occur at the membrane level.     

Digoxin quinidine interaction: a pharmacokinetic study in the isolated perfused rat liver

Digoxin-quinidine interaction was studied in the experimental model of isolated perfused rat liver. Neither digoxin nor quinidine were toxic to the isolated rat liver. The clearance of digoxin and quinidine by the liver was directly related to the rate of bile flow and the size of the initial dose of digoxin. In the presence of quinidine, after initial doses of digoxin of 0.5 and 1 micrograms, the concentration of digoxin in the perfusate was increased 2.5 and 3-fold. Its excretion in the bile was reduced by 45% and 20.5%, respectively (all comparisons, p less than 0.01). Digoxin concentration in the liver tissue was calculated and found to be appreciably elevated in the presence of quinidine. A reduction of about 30% (p less than 0.05) in the excretion of quinidine in the bile was observed in the presence of digoxin. Thus, a competition of digoxin and quinidine for biliary excretion was demonstrated as an underlying cause for digoxin-quinidine interaction in the isolated perfused rat liver.     

Effects of monensin on vesicular transport pathways in the perfused rat liver

In the rat hepatocyte, the internalization and degradation of asialoglycoproteins and the secretion of plasma and biliary proteins require specific intracellular sorting of vesicles. To aid in the biochemical characterization of these different vesicular pathways, we examined the effects of the ionophore monensin on the uptake and degradation of 125I-asialoorosomucoid (ASOR) and on the secretion of plasma and biliary proteins by the in situ perfused rat liver. In control livers, 77% of injected 125I-ASOR was extracted on first pass; 93% of the extracted radioactivity was released back into the circulation (totally degraded and some intact ASOR was found); and approximately 2% was recovered in the bile, some of which was intact. Monensin treatment decreased first pass uptake of 125I-ASOR to 57% and abruptly blocked the release of radioactivity into the perfusate and the bile. When hepatic proteins were biosynthetically labeled with 3H-leucine, monensin treatment dramatically reduced and delayed the secretion of newly synthesized proteins into both the perfusate and the bile. In contrast with control livers, in which secretion of protein into the perfusate preceded secretion of protein into the bile, TCA-precipitable 3H-protein appeared in bile about 20 min before TCA-precipitable 3H-protein appeared in the perfusate in monensin-treated livers. Thus, monensin treatment in the perfused liver blocked the degradation of asialoglycoproteins and inhibited the secretion of plasma proteins but had less effect on biliary protein secretion. These data document physiologic effects of monensin in an intact organ and suggest that biochemical distinctions between different vesicular pathways exist in the rat hepatocyte.     

Cholesterol synthesis in the perfused liver of pregnant hamsters

Pregnancy is a risk factor for the development of cholesterol gallstones. In pregnant women, biliary cholesterol saturation and secretion are increased. To investigate whether this was due to increased cholesterol synthesis, we studied hepatic cholesterol synthesis in Syrian Golden hamsters. Female controls and animals 10- to 14-days pregnant were studied. The studies were performed in the in situ perfused hamster liver. Cholesterol synthesis was determined by measuring the incorporation of 3H2O added to the perfusate into hepatic, perfusate, and bile cholesterol during a 90-min period. In both pregnant groups, bile flow decreased significantly, but biliary cholesterol concentration increased only in the 14-day pregnant group. The cholesterol synthesis rate averaged (mean +/- SD) 172 +/- 27, 127 +/- 37, and 552 +/- 79 nmol X hr-1 X g liver-1 in controls, 10-day, and 14-day pregnant animals, respectively. The 14-day pregnant animals secreted a markedly higher fraction (47.3 +/- 11.3 vs. 11.1 +/- 13.4%; P less than 0.01) of newly synthesized cholesterol into bile but not into perfusate. Chenodeoxycholate, but not cholate, synthesis rate was decreased in both pregnant groups. We conclude from our studies that hepatic cholesterol synthesis increases towards the end of pregnancy in the hamster and that more newly synthesized cholesterol is secreted into bile at that time. This could at least partially explain the increased biliary cholesterol saturation and secretion observed in women in the third trimenon, and explain pregnancy as a risk factor in the development of cholesterol gallstones.     

Effect of indomethacin on the uptake, metabolism and excretion of 3-oxocholic acid: studies in isolated hepatocytes and perfused rat liver

3 alpha-Hydroxysteroid dehydrogenase catalyzes the reduction of 3-oxo-bile acids and binds 3 alpha-hydroxy bile acids. Indomethacin is a competitive inhibitor of the enzyme. In incubations of isolated rat hepatocytes, indomethacin delayed the intracellular reduction and the initial uptake of 3-oxocholic acid. Following a tracer dose of 3-oxocholic acid in perfused rat liver, rapid biliary excretion was observed mainly as taurocholic acid. Only 1.1% of the dose was recovered in the caval outflow and nearly all appeared in the first 5 min collection. When the tracer dose was given after initiating a constant infusion of indomethacin (50 microM), a dramatic decrease in biliary excretion was observed, still mainly as taurocholic acid, and 14% of the dose was recovered in the caval effluent: 10% in the first 5 min collection, mainly as 3-oxocholic acid, followed by a steady, slow release of mainly taurocholic acid. The increased intrahepatic retention of bile acids and slow release into perfusate and bile in response to indomethacin are consistent with displacement of bile acids from cytosolic protein.     

The effect of chlordiazepoxide hydrochloride on the isolated perfused rat liver.

The effects of chlordiazepoxide-hydrochloride (CDZ) on the isolated perfused rat liver were examined. CDZ administration decreased bile flow, biliary excretion of sulfobromophthalein (BSP) and hepatic uptake of BSP. The addition of CDZ to the perfusate of livers obtained from phenobarbital (Pb) pretreated rats led to 50% greater reductions in bile flow, concentration of BSP in bile and hepatic uptake of BSP. The adverse effects of CDZ on BSP excretion per g liver, however, did not appear to be enhanced by Pb pretreatment. The complex nature of the interrelationship of the effects of Pb and of CDZ on the control liver prevented differentiation of the role of CDZ from that of a metabolite on the adverse effect on liver function.     

Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2)

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.     

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)     

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.     

Synthesis, intestinal absorption and metabolism of sarcosine conjugated ursodeoxycholic acid

Sarcosine conjugated ursodeoxycholic acid (SUDC) was synthesized and its intestinal absorption and metabolism were studied in rat and hamster. Intestinal absorption study using bile fistula rat shows that more than 90% of SUDC administered intraduodenally was excreted in the bile within 24 hr. No change of the administered bile acid was seen during the absorption from the intestine, the passage of the liver, and the excretion into the bile. When [24-14C]SUDC and [11,12-3H2]-ursodeoxycholic acid were administered orally to a hamster, more than 95% of both the administered 14C and 3H were recovered from the feces within 6 days. Most (77%) of the fecal 14C-labeled compound was SUDC, whereas 95% of the fecal 3H-labeled compound was unconjugated lithocholic acid. These results indicate that SUDC, unlike taurine or glycine conjugated bile acid, resists bacterial deconjugation and 7-dehydroxylation.     

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.     

Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions

Adult rat hepatocytes were maintained in culture for at least 1 month without losing the expression of their differentiated functions; they were cultured on lethally treated 3T3 fibroblasts inoculated at 35,000 cells/cm2 with medium containing 10-25 micrograms/ml hydrocortisone. Hepatocytes showed their typical morphology; they formed bile canaliculi, microvilli, and intercellular junctions with desmosomes and nexus; some formed structures that may resemble the perisinusoidal space of Disse. In addition, they showed DNA synthesis and expressed some liver-specific functions. They synthesized albumin and other proteins, which were exported to the culture medium. Like parenchymal liver cells in vivo, de novo fatty acid synthesis and esterification took place, and more than 80% of the lipids synthesized by the hepatocytes were secreted into the medium as triglycerides; they also showed cytochrome-P450 activity that was inducible with phenobarbital, suggesting that the hepatocytes have the capacity to metabolize drugs. These culture conditions allow the study of various hepatocyte differentiated functions, and they may provide the means to analyze the effect on liver of hormones, viruses and hepatotoxic chemicals and drugs; they may also indicate conditions adequate for serial growth of hepatocytes.     

LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.     

Biochemical site of regulation of bile acid biosynthesis in the rat

The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.     

Cholesterol and bile acid dynamics: comparative aspects

While the cholesterol concentration in a given tissue is similar in the rat, pig or man, the relative importance of the processes regulating the input (absorption and synthesis) and output (faecal cholesterol and bile acid excretions) of the cholesterol system is very different from one species to another. The rat, whose cholesterolaemia does not significantly increase after cholesterol addition to the diet ("hyporesponding" animal), successfully adapts its bile acid biosynthesis to variations in cholesterol input. This process accounts for 80 to 85% of cholesterol output, faecal cholesterol excretion being a minor process. The latter results from a low liver cholesterol secretion in the bile due to the low hydrophobicity of its main bile acids. Furthermore, in this animal a high intestinal synthesis of cholesterol and apolipoproteins (particularly B48) is observed. The latter are secreted as very light lipoproteins (chylomicrons and VLDL) with a faster plasma turnover than the VLDL (apoB100, E...) secreted by the liver. The "remnants" of rat VLDL are essentially very rapidly taken up by the liver; their interplasmatic transformation pathway into IDL and LDL is not very significant (less than or equal to 10%). Man, who has a more significant hypercholesterolaemia after exogenous cholesterol ingestion ("hyperresponding" subject) seems to have a less modulable capacity for transforming cholesterol into bile acids. This process accounts for only 50% of cholesterol output, faecal cholesterol excretion being quantitatively just as significant. Cholesterol concentration and the cholesterol/bile acid ratio are much higher in human than in rat bile, the main bile acids being more hydrophobic. While both the intestine and liver contribute to cholesterogenesis, the relative importance of the latter is probably greater in man than in the rat. Moreover, a larger fraction of plasma VLDL is transformed into IDL and LDL, the latter representing the main plasma cholesterol carrier. Determining whether the differences between the biodynamics of cholesterol processes in the rat and in man can be generalised to mammals with low or high sensitivities to hypercholesterolaemia and atherosclerosis seems to be a fundamental research objective for the next few years.