Modulating effects of bile salt hydrophobicity on bile secretion of the major protein of the bile lipoprotein complex

Bile lipids are secreted in association with a newly identified major apoprotein called anionic polypeptide fraction-calcium binding protein (APF-CBP), which is synthesized in the hepatocytes and has been detected in both bile and plasma and characterized. The secretion of the lipids in bile depends both on the concentration and the hydrophobicity of the bile salts (BS) secreted. The present study was undertaken to determine whether the synthesis and the secretion of APF-CBP are similarly regulated by BS, using two methods. The synthesis and secretion of labelled, newly synthesized APF-CBP by isolated rat hepatocytes were monitored by solid-phase immunoassay. For this purpose, hepatocytes were incubated with either glycodeoxycholate (GDC) or taurocholate (TC). The synthesis and secretion of labelled, newly synthesized APF-CBP by perfused rat liver were measured by immunological enzyme-linked assay (ELISA) upon perfusing the liver with either GDC or TC. We found that (i) the synthesis and the secretion of APF-CBP were increased during either TC or GDC perfusion, but the increase was more pronounced with TC; (ii) in GDC perfusion the APF-CBP levels measured were more closely related to the levels of bile salts and not to phospholipid levels, (iii) when the two bile salts were perfused in reverse order,i.e., first GDC and then TC, the secretion of APF-CBP in bile decreased when GDC was perfused, but increased when TC was perfused. Similar results were obtained in experiments with isolated hepatocytes. The data suggest that the hydrophobicity of the BS used in the infusion modulates the synthesis and secretion of APF-CBP. In the liver, the pool of APF-CBP can be modified by BS and responds rapidly to BS stimulation.     

Call for naminations award in lipid chemistry

Summary It has been claimed that the hepatic passage of conjugated bile acids does not increase oxygen uptake by the isolated perfused rat liver. We studied a variety of bile acids in the single-pass perfused rat liver. All bile acids studied except taurocholate and tauro-β-muricholate induced an increase in oxygen uptake of 2.1%–11.2% (baseline 2.54±0.57 μmoles/min per g liver). Raised values for oxygen uptake were not specifically associated with the critical micellar concentration of the bile acid, with bile acid uptake, or with bile acid secretion. It is postulated that bile acid toxicity may have an intracellular effect on hepatic oxygen uptake. Supported by NIH grant, no. AM 16/10 of the US Public Health Service (Dr. Miyai). J. S. was a recipient of a grant from the Thyssen Stiftung, FRG. Part of this paper was presented at the EASL meeting in Leuven, Belgium, 1988     

Dietary fat alters biliary lipid secretion in the hamster

Dietary fat has been found to alter the incidence of cholesterol gallstones in hamsters: butterfat intensifies while safflower oil reduces lithiasis. We now report how dietary fat affects bile flow and biliary lipid secretion in this model. Male hamsters were fed one of three experimental diets: a control diet (containing 0.3% cholesterol); control diet +4.0% butterfat; or control diet +4.0% safflower oil. After three weeks, bile samples were collected via an external biliary fistula. The endogenous bile acid pool was depleted for 120 min followed by increasing rates of taurocholate infusion for 160 min. Basal secretion of biliary lipids was measured during the bile acid depletion period. Basal bile flow and bile acid output were not significantly different in the three groups. Dietary butterfat increased basal cholesterol output compared to the control diet (0.037 vs. 0.025 μmol/min·kg, respectively); safflower oil did not change cholesterol output (0.027 μmol/min·kg). Hamsters fed butterfat or safflower oil secreted more phospholipid (0.171 and 0.178 μmol/min·kg, respectively) than controls (0.131 μmol/min·kg). The cholesterol/phospholipid output ratio of the butterfat group was higher than the safflower oil group (0.220 vs. 0.153, respectively). Effects of dietary fat on several relationships between bile flow and biliary lipid secretion were analyzed by linear regression using the data for the entire bile collection period (bile acid depletion and taurocholate infusion). Butterfat and safflower oil did not change either bile acid dependent or bile acid independent bile flow. Hamsters fed butterfat had a higher linkage coefficient (slope) of cholesterol vs. bile acid output than the safflower oil group (0.023 vs. 0.009, respectively). The linkage coefficient of phospholipid vs. bile acid output of the butterfat group was higher than the controls (0.278 vs. 0.185, respectively). In summary, butterfat induced a high cholesterol and phospholipid secretion with a high cholesterol/phospholipid output ratio; safflower oil induced a high phospholipid secretion with a low cholesterol/phospholipid output ratio. Butterfat and safflower oil have different effects on biliary lipid secretion. These differences in biliary lipid secretion may explain, in part, how butterfat and safflower oil differ in affecting gallstone formation in hamsters.     

Effects of bile salts on rat hepatic acyl CoA:Cholesterol acyltransferase

Acyl CoA:cholesterol acyltransferase (EC2.3.1.26, ACAT), responsible for intracellular esterification of cholesterol, may play an important role in cholesterol trafficking within the cell, and thus, in maintenance of cellular cholesterol homeostasis. Bile acids are potential regulators of cholesterol trafficking in the liver. Therefore, the effect of bile salts on hepatic ACAT activity was studied in the perfused rat liver. ACAT activity was increased after liver perfusion with either taurocholate or taurochenodeoxycholate. However, addition of these bile salts at physiological concentrationsin vitro had little effect on microsomal ACAT activity. The increase in hepatic ACAT activity due to perfusion with bile salts was accompanied by reduced accumulation of very low density lipoprotein cholesterol in the perfusate, but there was no effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Hepatic ACAT activity was decreased after bile diversion for four hours in the intact animal. This treatment had no statistically significant effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. These data suggest that bile salts induce changes in hepatic compartmentation and traffic of cholesterol within the hepatocyte accompanied by response of ACAT activity to maintain cellular cholesterol homeostasis.     

Nonspecific lipid transfer proteins as probes of membrane structure and function

A protein that accelerates transfer of phospholipids of varying head group and fatty acid composition has been purified from bovine liver. As previously found for other phospholipid transfer proteins, “nonspecific lipid transfer protein” stimulates a kinetically biphasic transfer of radioactively labeled phospholipid from small unilamellar vesicles to unlabeled multilamellar vesicles. The kinetics are consistent with rapid transfer of phospholipid from the outer monalyer and slow transfer of that localized in the inner monolayer (half-times greater than 3 days for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). Protein catalyzed transfer is inhibited by high ionic strength and has an activation energy of 35 kJ/mol. The broad lipid specificity and ease of large-scale purification make these proteins candidates for membrane phospholipid compositional modification. The compositions of rat liver mitochondrial and microsomal membranes and Morris hepatoma 7288c mitochondrial membranes were altered by incubation with lipid vesicles and nonspecific lipid transfer protein. Incubation with phosphatidylcholine vesicles led to increased levels of phosphatidylcholine and decreased levels of other transferrable lipids (phosphatidylethanolamine, phosphatidylinositol, and cholesterol) unless the latter were included in the vesicles. When vesicles containing dipalmitoylphosphatidylcholine were incubated with microsomal membranes, a large increase in disaturated phosphatidylcholine was also observed. These changes in composition were correlated with activities of membrane enzymes. It appears that microsomal glucose-6-phosphatase is inhibited by increased phosphatidylcholine saturation. Moreover, this enzyme is also inhibited by decreases in the phosphatidylethanolamine/phosphatidylcholine ratio whereas NADPH cytochrome c reductase is not. Likewise, decreased cholesterol to phospholipid ratios did not greatly affect the abnormally low levels of hepatoma succinate cytochrome c reductase activity. This paper was presented at the 73rd AOCS annual meeting, Toronto, Canada, May 1982.     

Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver

The effect of various physicochemical forms of substrate on the activity of acid cholesteryl ester hydrolase isolated from rat liver lysosomes was studied. The amount of sodium taurocholate was varied in the substrate mixture which contained constant amounts of egg phosphatidylcholine (PC) and cholesteryl oleate. The resulting substrate forms produced were PC vesicles, PC vesicles with incorporated sodium taurocholate, mixed micelles, and mixed micelles together with free bile salt micelles. Gradually increasing amounts of sodium taurocholate activated cholesteryl oleate hydrolysis until the molar sodium taurocholate/PC ratio of ca. 0.6; thereafter hydrolytic activity decreased rapidly. The presence of sodium taurocholate micelles clearly inhibits cholesteryl oleate hydrolysis. We therefore propose that the activation observed at low bile salt concentrations depends on bile salt interaction with the substrate vehicle, whereas the inhibition observed at high bile salt concentrations depends on sodium taurocholate interacting with the enzyme. When comparing different phospholipid components in the supersubstrate, the enzyme activity was highest in the presence of dioleyl PC and decreased when present with dipalmitoyl PC and egg PC. Egg lysoPC completely inhibited the enzyme activity. A net negative charge on the surface of the vesicle substrate increased cholesteryl ester hydrolase activity while a net positive charge on the surface inhibited the enzyme activity. Only part of the product inhibition of cholesteryl oleate hydrolase caused by Na-oleate was reversible when tested with bovine serum albumin present in the incubation mixture.     

Effects of Aramchol on in vitro bile cholesterol crystallization and bile acid detergency

The hydrophilic bile acid ursodeoxycholic acid may dissolve cholesterol gallstones and is beneficial in cholestatic liver diseases. The C20 fatty acid‐bile acid conjugate arachidyl amido cholanoic acid (Aramchol) could be a more effective option. We therefore studied its effects on cholesterol crystallization and on bile salt‐induced cytotoxicity. Effects of Aramchol at therapeutically relevant concentrations on crystallization in supersaturated model biles (by microscopy and chemical measurement), on the ternary cholesterol‐taurocholate‐phosphatidylcholine phase diagram, and on micelle ? vesicle transitions (by serial dilution or by incubation of cholesterol‐phosphatidylcholine vesicles with taurocholate) were evaluated. Effects on bile salt‐induced cytotoxicity were determined in erythrocytes and CaCo2 cells. Incorporation of Aramchol in model biles did not change micellar cholesterol solubilization, induced a small rightward shift of crystal‐containing zones of the ternary phase diagram, exerted no appreciable effects on vesicle ? micelle transitions and had only minor effects on cholesterol crystallization. Bile salt‐induced cytotoxicity was increased by Aramchol in all models. Since Aramchol does not affect cholesterol crystallization, its previously reported beneficial effects in animal gallstone models should relate to other mechanisms. Since Aramchol increases bile salt detergency, it is not likely to be beneficial in cholestatic liver disease.     

Effects of oleic,arachidonic and 5,8,11,14-nonadecatetraenoic acids on lipid secretion and ketogenesis in perfused rat liver

The effects of perfused oleic (18∶1n−9), arachidonic (20∶4n−6) and 5,8,11,14-nonadecatetraenoic (19∶4n−5) acids on triglyceride and cholesterol secretion and ketone body production were studied in isolated rat liver. As compared to oleic and 19∶4n−5 acids, both ketone body production and triglyceride secretion were significantly lowered when arachidonic acid was perfused. The concentration of triglyceride in the post-perfused liver was lower upon perfusion with arachidonic acid than upon perfusion with oleic acid or 19∶4n−5 acid. Cholesterol secretion in the liver perfused with arachidonic acid or 19∶4n−5 acid was significantly higher than with oleic acid. The concentration of cholesterol in the post-perfused liver was slightly but significantly higher with 19∶4n−5 acid than with the other fatty acids. The results suggest that 19∶4n−5 acid when compared with arachidonic acid affects lipid metabolism in liver differently.     

Bile acid metabolism in mammals: VII. Studies on sex differences in deoxycholic acid metabolism in isolated perfused rat liver

The hepatic metabolism of deoxycholic acid was studied using the isolated perfused rat liver technique. In 20 perfusions, 10 involving the livers of male rats and 10 involving the livers of female rats, 30 μmoles deoxycholic acid was added to the perfusion medium. In 10 perfusions, 5 male and 5 female, 1 μmole deoxycholic acid was added to the perfusion medium. In 10 of the high dose studies and in the 10 low dose studies, 1 μCi deoxycholic acid-C-24-C¹⁴ also was added. The deoxycholic acid was added to 100 ml perfusion medium after 2 hr of baseline perfusion, and the studies were continued another 3 hr. Biliary bile acids were analyzed by combined thin layer and gas chromatography, and the radioactivity content of the perfusion medium and liver was documented. Although there was no sex difference in total bile acid secretion in the high dose studies, there were sex differences in the bile acid secretion rate and in the quantitative secretion of individual bile acids. The biliary secretion of deoxycholic acid and cholic acid was immediate in the female studies and delayed in the male, and the amounts of cholic acid and sulfated deoxycholyl-taurine secreted were considerably greater in the male studies. In the low dose studies the isolated perfused liver of the female rat converted more deoxycholic acid to cholyl-taurine than did that of the male rat. There are sex differences in the hepatic metabolism of deoxycholic acid. In contrast to those found in the case of chenodeoxycholic acid, these sex differences are not impressive when physiological amounts of deoxycholic acid are presented to the liver.     

Cholesterol secretion from hepatocytes induced by triacylglycerol and apolipoprotein E

The mechanism for the increase in plasma cholesterol levels in cholesterol-fed rats following chylomicron transport was investigated in intact animals, in isolated perfused liver, and in hepatocytes in monolayer cultures. Intravenous administration of egg phosphatidylcholine in amounts greater than those required to cause a plasma cholesterol response when given as chylomicrons was without effect. This makes it unlikely that increased plasma cholesterol levels resulted from the recruitment of tissue cholesterol by the plasma chylomicron phospholipids that persisted in the plasma after triacylglycerol clearance. The hepatic origin of the increased plasma cholesterol levels was directly confirmed by two hepatic perfusion experiments. When cholesterol-fed rats received intravenous chylomicrons prior to isolated hepatic perfusion, more cholesterol was secreted by the liver than when the rats were injected intravenously with buffer. Perfusion of apolipoprotein E (apo E)-rich triacylglycerol emulsions through the livers also enhanced cholesterol secretion. The increase in hepatocyte cholesterol secretion seen with cholesterol-fed rats was also noted in monolayer cultures following incubation with apo E rich-triacylglycerol emulsions. The apolipoprotein or the emulsion alone, or apo E-rich phosphatidylcholine liposomes, had no effect. The data confirm previous indirect observations that the liver is the source of cholesterol that appears in plasma following transport of chylomicrons or following a lipid-rich meal in cholesterol-fed rats. The data also re-emphasize the importance of providing apo E with triacylglycerol emulsions to initiate secretion of lower density lipoproteins by the liver.     

Effects of lysophosphatidylcholine on jejunal water and solute transport in the rat in vivo

The effects of lysophosphatidylcholine on jejunal water and solute transport were studied in vivo in the rat. Five mM lysophosphatidylcholine significantly reduced absorption of water, electrolytes and glucose (P<0.05) and 10 mM lysophosphatidylcholine induced net fluid secretion. The effects of 10 mM lysophosphatidylcholine were significantly reduced in the presence of 5 mM phosphatidylcholine (P<0.05) and 2 mM cholesterol (P<0.05). The fractional absorption of lysophosphatidylcholine decreased with increasing concentration of the detergent in the perfusion solution. Increasing concentrations of taurocholate in the perfusion solutions potentiated the effects of lysophosphatidylcholine (P<0.01), although 10 mM taurocholate by itself had no significant effect on intestinal water and electrolyte transport. The data establish that lysophosphatidylcholine, a zwitterionic detergent, affects intestinal transport in the same way as bile acids, fatty acids and synthetic cationic or nonionic detergents. By comparison with the response of the human jejunum to taurodeoxycholate, it is likely that lysophosphatidylcholine generated during the normal process of digestion has an effect on intestinal water and solute transport in man.     

Mechanisms of linoleic acid uptake by rabbit small intestinal brush border membrane vesicles

We examined the initial transport of a long-chain unsaturated fatty acid, linoleic acid, by brush border membrane vesicles isolated from rabbit small intestine. This preparation allowed us to examine the transport of linoleic acid across the brush border membrane without the effect of the unstirred water layer or cytosol binding proteins. Linoleic acid was solubilized in a 2 mM taurocholate solution which did not compromise the functional integrity of the vesicles. Linoleic acid uptake in the range of 1 to 100 μM followed passive diffusion kinetics. Time course study showed that linoleic acid uptake reached maximal levels during the initial 15 seconds. Although the amount of linoleic acid accumulated in the vesicles diminished over the next 30 minutes, the molar quantity was still twentyfold higher than that of D-glucose (6.5 vs 0.33 nmol/mg protein). Uptake of D-glucose by the vesicles demonstrated typical osmotic responsiveness. We found no osmotic effect on linoleic acid uptake. Hypotonic lysis of membrane vesicles loaded with linoleic acid released 40% of the fatty acid. We concluded that a major portion of the accumulated fatty acid was bound to or incorporated into the membrane itself while ca. 40% did traverse the membrane and accumulated in the intravesicular space as nonmicellar aggregates. The known inhibitors of anion transport, diisothiocyanatostilbene and isothiocyanatostilbene did not change the transport of linoleic acid. We conclude that, in the absence of an unstirred layer or cytosol proteins, linoleic acid transport at up to 100 μM concentration is passive with rapid accumulation both by the cell membrane and the lumen of vesicles.     

Metabolism of n−3 polyunsaturated fatty acids by the isolated perfused rat liver

The hepatic metabolism of oleic acid and n−3 fatty acids (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and secretion of very low density lipoprotein (VLDL) were studied in isolated perfused rat livers from normal chow fed male rats. The basal perfusion medium contained 30% bovine erythrocytes, 6% bovine serum albumin (BSA), and 100 mg/dL glucose, in Krebs-Henseleit bicarbonate buffer (pH 7.4) which was recycled through the liver for 2 hr. Individual fatty acids (EPA, DHA or oleic acid), as complexes with 6% BSA, or albumin alone, were infused at a rate of 70 μmol/hr. When any of these fatty acids was infused at this rate, the ambient concentration in the medium was maintained at 0.3–0.4 μmol/mL, indicative of similar hepatic rates of uptake for each fatty acid (i.e., approximately 6 μmol/g liver/hr). When fatty acid was not infused, the ambient free fatty acid level was 0.16 μmol/mL. The concentrations of infused free fatty acids increased appropriately in the perfusion medium; however, with infusion of EPA, DHA, or oleate, the concentrations of perfusate palmitate and linoleate were the same as when fatty acid was not infused. Additionally, the perfusate concentration of oleate in the free fatty acid fraction was not affected by infusion of EPA and DHA. These data indicate a constant outflow of endogenous fatty acid unaffected by the presence of the exogenously supplied fatty acid. The net secretion rate of VLDL lipids and protein was stimulated by infusion of oleate, whereas when EPA was infused, secretion rates were lower and similar [except for VLDL cholesterol (C), which was greater] to those occuring when fatty acid was not provided. DHA stimulated the secretion of VLDL triacylglycerol (TG), phospholipid (PL) and C to a similar rate, as did oleate, but secretion of VLDL cholesteryl ester (CE) and protein was lower and similar to that with EPA. VLDL and hepatic TG and CE were enriched with the infused fatty acids, compared to experiments without fatty acids, as determined by gas chromatography. Enrichment of PL, however, was significant only in liver upon infusion of EPA. The formation of¹⁴CO₂ and perchloric acid soluble products from [1-¹⁴C]EPA, considered separately, did not differ statistically from that obtained with [1-¹⁴C]oleate, although the mean values were higher with [1-¹⁴C]EPA. However, the sum of oxidation products derived from EPA was significantly greater than that from oleate. Incorporation of [1-¹⁴C]EPA into TG and CE, but not into PL, was lower as compared to that from [1-¹⁴C]oleate. These lower rates of incorporation of [1-¹⁴C]EPA into VLDL lipids therefore paralleled the mass fatty acid enrichment-patterns. It may be concluded that EPA is used to a similar extent as oleate for synthesis of PL, but is a poorer substrate for synthesis of TG. The reduced output of newly synthesized (radioactive) PL reflected the lower hepatic output of VLDL. Since hepatic uptake of EPA, DHA or oleate was identical, utilization of EPA for TG synthesis was less than that of oleate or DHA. Further-more, utilization of endogenous fatty acids for TG synthesis and secretion of the VLDL was reduced in the presence of EPA. The decreased TG synthesis resulted in reduced formation of VLDL for transport of TG from the liver. These effects taken together with an apparently increased oxidation of EPA provide substantial evidence for a decrease in formation of VLDL and transport of TG, PL, C and CE into the circulation in response to EPA. DHA, however, appears to be an adequate substrate for TG synthesis and stimulates VLDL secretion. The reduced transport of CE may reflect lower selectivity of DHA by acyl-CoA; cholesterol acyltransferase for CE formation.     

The effect of lipids on taurocholate absorption from intestinal loops in the rat

The rates of uptake and serosal trans-fer of [¹⁴C]-labelled taurocholate (7.77 mM in bicarbonate buffer, pH 6.5) were determined in situ in ligated segments of rat intestine in the presence of lipids. Oleic acid, monoolein, lecithin, and lysolecithin enhanced taurocholate uptake and transfer in the jejunum, each lipid exhibiting and optimal concentration at which the bile acid fluxes were maximal. The maximal rates of bile acid uptake observed with the various lipids were close to four times the uptake rates found with the lipid-free taurocholate medium, whereas serosal transfer rates under optimal conditions were enhanced about six-fold. The optimal concentrations differed widely among the various lipids, being inversely related to the lipids’ polarity. Simultaneous measurement of taurocholate and [³H]-labelled oleic acid showed that under optimal conditions, when the molar concentration of oleic acid was about equal to that of the bile acid, the fatty aicd and bile acid also exhibited closely similar rates of absorption. At other fatty acid concentrations, the fractional rate of absorption of the bile acid was much lower than that of the fatty acid. The rates of uptake and serosal transfer of pure taurocholate by the ileum exceeded those of the jejunum by factors of about 7 and 15, respectively, but in the presence of lipids this difference in absorptive capacity for bile acid between the distal and proximal segment largely disappeared.     

Arachidonic acid intestinal absorption: Mechanism of transport and influence of luminal factors on absorption in vitro

The mechanism and characteristics of intestinal absorption of arachidonic acid were studied in vitro using everted intestinal sacs of the rat. Arachidonic acid absorption was studied at concentrations of 5 μM to 8.36 mM. The plot of absorption rate vs. concentration fitted best to a rectangular hyperbola at low μM concentrations and to a straight linear relationship in the mM range of concentrations. Metabolic inhibitors and uncouplers did not change absorption in either range of concentrations. The absorption of arachidonic acid increased with thinning of the unstirred water-layer, decrease in the pH, or the substitution of sodium taurocholate by Pluronic F 68 or Tween 80. Absorption decreased following the equimolar additions of oleic, linoleic, and linolenic acids. Absorption rate did not change when the taurocholate concentration was varied from 5–15 mM or following the additions of butyric or glutamic acids, leucine, lysine, or dextrose. It was concluded that arachidonic acid is absorbed by a concentration-dependent dual mechanism of transport which is not energy dependent. At the low μM range of concentrations, facilitated diffusion is predominant, while at mM concentrations, simple diffusion is the dominant mechanism of absorption. Changes in the intestinal fluid composition, flow rate, and pH can modify the rate of absorption of arachidonic acid.     

Inhibition and induction of bile acid synthesis by ketoconazole effects on bile formation in the rat

The effects of ketoconazole, an antimycotic agent, and metyrapone, an inhibitor of mixed function oxidases, on bile acid synthesis were compared in the rat bothin vitro andin vivo. In rat liver microsomes, ketoconazole was much more potent than metyrapone in inhibiting the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acids. The I₅₀ values were 0.42 μM and 0.91 mM for ketoconazole and metyrapone, respectively. Intraduodenal administration of ketoconazole caused a rapid, dose-dependent reduction of bile acid synthesis in eight-day bile diverted rats. A single dose of 50 mg/kg reduced bile acid synthesis to 5% of control value; the same dose of metyrapone caused a reduction to only 85%. Inhibition of bile acid synthesis by ketoconazole was followed by a marked overshoot. At 28 hr after injection of 50 mg/kg of the drug, formation of bile acids was stimulated maximally by 45% compared to control value and remained elevated for more than 20 hr thereafter. Synthesis of all primary bile acids was affected to the same extent. Cholesterol 7α-hydroxylase activity in livers of ketoconazole treated (30 mg/kg) rats with an intact enterohepatic circulation was increased by 70% at 16 hr after i.p. injection of the drug. During the very large decrease of biliary bile acid output with ketoconazole, bile flow rate was relatively increased, due to stimulation of the bile acid-independent fraction of bile flow. The latter effect can probably be explained as caused by biliary secretion of osmotically active metabolites of ketoconazole.     

The incorporation of fatty acids of different chain length into liver and biliary lipids in the perfused rat liver

In an attempt to correlate the incorporation of fatty acids (FA) of different chain length into liver and biliary lipids’ isolated rat livers were perfused for 2 h with Krebs-Ringer bicarbonate containing 1% albumin and 10 μmol of [1-¹⁴C]-labeled FA: C₂’ C₈’ C₁₀’ C₁₂’ C₁₆’ and C_18∶1. One to 1.36 μmol of medium-chain fatty acids (MCFA’ C₈’ C₁₀’ and C₁₂) and 6.6 μmol of long-chain FA (LCFA) were incorporated into liver lipids’ 40% of the latter into phosphatidylcholine (PC). ¹⁴C-acetate (13 nmol) was incorporated into biliary cholesterol; ¹⁴C-MCFA contributed only 3.2–5 nmol; LCFA did not lead to newly synthesized cholesterol. Newly synthesized liver PC (2.75 to 3.25%) and newly synthesized liver cholesterol (6.5 to 10%) were secreted into bile. The specific radioactivity of biliary PC after infusion of all-saturated FA was 3.8–6.8 times higher than that of liver PC; for C_18∶1 it was only 1.7-fold. The specific radioactivity of biliary cholesterol’ as compared to liver cholesterol’ was 12 times higher for C₂ and five times higher for MCFA. This indicates that a considerable proportion of the newly synthesized lipids was secreted into bile prior to significant mixing with preexisting liver PC and cholesterol pools. liver PC contained 8% of unchanged ¹⁴C−C₁₂; while ¹⁴C−C₁₀ was not detected. Biliary PC’ in contrast’ contained 18% of unchanged ¹⁴C−C₁₂ and 3% ¹⁴C−C₁₀. These results suggest that after prolonged infusion of medium-chain triacylglycerols/longchain triacylglycerols to patients’ biliary PC may become enriched with MCFA. In addition’ the oxidation of these FA may provide C-2 units which increase cholesterol synthesis.     

Hepatic bile acid elution by albumin and bile acid content in isolated rat hepatocytes

Bile acid contents were determined for isolated rat hepatocytes. During the course of isolating the hepatocytes, perfusion of rat liver with buffer containing 2% albumin eluted a significant amount of bile acids. The elution was proportional to the volume of the buffer and attributable to albumin in the buffer. The isolated hepatocytes prepared by perfusion with 0.1% albumin buffer, which eluted a negligible amount of bile acids, contained 95±12 μg/10⁸ cells of bile acids. The major bile acids were cholic acid (22%), β-muricholic acid (34%) and hyodeoxycholic acid (10%). Levels of the other bile acids were less than 3%. Peak 8, unidentified but presumed to be a trihydroxycholanoic acid, accounted for 19%.     

The cholesterol-lowering effect of guar gum in rats is not accompanied by an interruption of bile acid cycling

A viscous hydrocolloid (guar gum, GG; 2.5% of the diet) or a steroid sequestrant (cholestyramine; 0.5% of the diet) was included in semipurified diets containing 0.2% cholesterol to compare the cholesterol-lowering effects of each agent in rats. In the present model, GG significantly lowered plasma cholesterol (−25%), especially in the density <1.040 kg/L fraction, whereas cholestyramine was less potent. Bile acid fecal excretion significantly increased only in rats fed cholestyramine, similar to the cecal bile acid pool; the biliary bile acid secretion was accelerated by GG, but not their fecal excretion, whereas GG effectively enhanced neutral sterol excretion. As a result, the total steroid balance (+13 μmol/d in the control) was shifted toward negative values in rats fed the GG or cholestyramine diets (−27 or −50 μmol/d, respectively). Both agents induced liver 3-hydroxy-3-methylglutaryl-CoA reductase, but cholestyramine was more potent than GG in this respect. The present data suggest that, at a relative low dose in the diet, GG may be more effective than cholestyramine in lowering plasma cholesterol by impairing cholesterol absorption and by accelerating the small intestine/liver cycling of bile acids, which is interestingly, accompanied by reduction of bile acid concentration in the large intestine.     

Action of three bile acids on hepatic and intestinal cholesterogenesis in the rat

Incorporation of [1¹⁴−C] acetate into cholesterol by subcellular particles from the liver and the small intestine of rats with a biliary diversion and a duodenal perfusion of sodium taurocholate, taurochenodeoxycholate or taurodehydrocholate, was studied in vitro. In the liver, taurochenodeoxycholate prevented the increase of cholesterol synthesis induced by biliary drainage. Taurocholate had no action on cholesterol synthesis at any time, day or night. Intestinal synthesis of cholesterol was reduced by taurocholate and taurochenodeoxycholate but was not modified by taurodehydrocholate infusion.