The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes was studied. Mevalonolactone was taken up by the cells and converted to cholesterol, cholesterol ester and tauroconjugates of bile acids. The addition of mevalonolactone had little effect on the conversion of endogenous cholesterol to taurocholic acid; however, taurochenodeoxycholic acid synthesis was stimulated. 25-30% of the cholesterol synthesized from mevalonolactone was converted to taurochenodeoxycholic, taurocholic and two so-far unidentified bile acids. All bile acids were secreted into the incubation medium. When cholesterol was added as mixed liposomes with phosphatidylcholine, it was taken up by the cells and converted to bile acids. At low concentrations of liposomes, the greater part of the cholesterol which was taken up by the cells was converted to bile acids. At higher concentrations, considerable amounts of cholesterol and cholesterol ester accumulated inside the cells. When mevalonolactone and cholesterol liposomes was added together, both substrates were used simultaneously for bile acids synthesis. HDL cholesterol was the best substrate tested, yielding large amounts of two, so-far, unidentified bile acids (possibly allo-bile acids) and smaller amounts of taurocholic and taurochenodeoxycholic acid. Addition of HDL suppressed the conversion of endogenous cholesterol to taurocholic acid; taurochenodeoxycholic acid synthesis, however, was stimulated.     

Effects of ursodeoxycholic acid, analogues of ursodeoxycholic acid and combination of bile acids on bile acid synthesis in cultured rat hepatocytes

The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes. Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA). Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback. Each individual steroid was added to the culture media at concentrations ranging from 10 to 200 microM. Mixtures of taurochenodeoxycholate (TDCA) and taurocholate in concentrations ranging from 150 to 600 microM alone and in combination with phosphatidylcholine (10-125 microM) and cholesterol (3-13 microM) were also tested for their effects on bile acid synthesis. Rates of bile acid synthesis were determined as the conversion of added lipoprotein [4-14C]cholesterol or [2-14C]mevalonate into 14C-labeled bile acids and by GLC quantitation of bile acids secreted into the culture media. Individual bile acids, bile acid analogues, combination of bile acids and mixture of bile acids with phosphatidylcholine and cholesterol failed to inhibit bile acid synthesis in cultured hepatocytes. The addition of UDCA or UCA to the culture medium resulted in a marked increase in the intracellular level of both bile acids, and in the case of UDCA there was a 4-fold increase in beta-muricholate. These results demonstrate effective uptake and metabolism of these bile acids by the rat hepatocytes. UDCA, UCA, TUDCA and GUDCA also failed to inhibit cholesterol-7 alpha-hydroxylase activity in microsomes prepared from cholestyramine-fed rats. The current data confirm and extend our previous observations that, under conditions employed, neither single bile acid nor a mixture of bile acids with or without phosphatidylcholine and cholesterol inhibits bile acid synthesis in primary rat hepatocyte cultures. We postulate that mechanisms other than a direct effect of bile acids on cholesterol-7 alpha-hydroxylase might play a role in the regulation of bile acid synthesis.     

Group separation of bile acids and salts by silicic acid column chromatography

Group separations of unconjugated and conjugated bile acids and salts were performed using mixtures of conventional solvents by chromatography on columns of silicic acid. The results suggest that this method is useful for group separations of mono-, di-, and trihydroxycholan-24-oic acids and their conjugates with good recoveries. This method is advantageous for synthesis work, especially for the purification of conjugated and sulfated bile acids and salts, and is applicable for the group separation of glycine and taurine conjugates. The application of this method to human gallbladder bile salts is demonstrated.     

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.     

EP4 emerges as a novel regulator of bile acid synthesis and its activation protects against hypercholesterolemia

Prostaglandin E receptor subtype 4 (EP4) knockout mice develops spontaneous hypercholesterolemia but the detailed mechanisms by which EP4 affects cholesterol homeostasis remains unexplored. We sought to determine the cause of hypercholesterolemia in EP4 knockout mice, focusing on the role of EP4 in regulating the synthesis and elimination of cholesterol. Deficiency of EP4 significantly decreased total bile acid levels in the liver by 26.2% and the fecal bile acid content by 27.6% as compared to wild type littermates, indicating that the absence of EP4 decreased hepatic bile acid synthesis and their subsequent excretion in stools. EP4 deficiency negatively regulate bile acid synthesis through repression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK)-mediated cholesterol 7α-hydroxylase (CYP7A1) expression and that the hypercholesterolemia in EP4 knockout mice is due to a defect in cholesterol conversion into bile acids. Deficiency of EP4 also increased de novo cholesterol synthesis and altered cholesterol fluxes in and out of the liver. Treating high fat diet-challenged mice with the pharmacological EP4 agonist, CAY10580 (200?μg/kg body weight/day i.p) for three weeks effectively prevented diet-induced hypercholesterolemia, enhanced endogenous bile acid synthesis and their fecal excretion. In summary, EP4 plays a critical role in maintaining cholesterol homeostasis by regulating the synthesis and elimination of bile acids. Activation of EP4 serves as an effective novel strategy to promote cholesterol disposal in the forms of bile acids in order to lower plasma cholesterol levels.     

Synthesis of new bile acid analogues and their metabolism in the hamster: 3 alpha, 6 alpha-dihydroxy-6 beta-methyl-5 beta-cholanoic acid and 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid

This paper reports the chemical synthesis of two new bile acid analogues, namely, 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid from 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acid and describes their metabolism in the hamster. A Grignard reaction of the oxo acid with methyl magnesium iodide in tetrahydrofuran gave two epimeric dihydroxy-6-methyl-cholanoic acids which were separated as the methyl esters by silica gel column chromatography. The configuration of the 6-methyl groups was assigned by proton nuclear magnetic resonance spectroscopy and was supported by the chromatographic properties of the new compounds. The metabolism of the two new bile acid analogues was studied in the hamster. After intraduodenal administration of the 14C-labeled analogues into bile fistula hamsters, both compounds were absorbed rapidly from the intestine and secreted into bile. Intravenous infusion studies revealed that these compounds were efficiently extracted by the liver; the administered analogues became major biliary bile acids, present as either the glycine or taurine conjugates. These compounds are useful to study the effect of methyl-substituted bile acids on cholesterol and bile acid metabolism and may possibly possess cholelitholytic properties.     

Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis

The in vivo role of the nuclear receptor SHP in feedback regulation of bile acid synthesis was examined. Loss of SHP in mice caused abnormal accumulation and increased synthesis of bile acids due to derepression of rate-limiting CYP7A1 and CYP8B1 hydroxylase enzymes in the biosynthetic pathway. Dietary bile acids induced liver damage and restored feedback regulation. A synthetic agonist of the nuclear receptor FXR was not hepatotoxic and had no regulatory effects. Reduction of the bile acid pool with cholestyramine enhanced CYP7A1 and CYP8B1 expression. We conclude that input from three negative regulatory pathways controls bile acid synthesis. One is mediated by SHP, and two are SHP independent and invoked by liver damage and changes in bile acid pool size.     

Feedback inhibition of bile acid synthesis in cultured pig hepatocytes

Bile acid synthesis by cultured pig hepatocytes, as measured by conversion of [14C]cholesterol to bile acids, increased during the second and third day of culture. This rise was inhibited after addition of various conjugated and unconjugated bile acids in a concentration of 100 microM. It could be completely prevented by cycloheximide, indicating that de novo protein synthesis is required for the increase in bile acid formation. No effect of exogenous bile salts on LDH release to the medium or on cellular ATP content was observed, demonstrating that hepatocyte viability was not affected. During the period in which bile acid synthesis was inhibited, pig hepatocytes were able to accumulate taurocholic acid (100 microM) up to 7-18 nmol per mg cell protein (decreasing during culture time). It is concluded that feedback regulation of bile acid synthesis is exerted by direct action of bile acids on the hepatocyte.     

Hepatocyte-hepatoma cell hybrids. Characterization and demonstration of bile acid synthesis

Hybrids were created by fusion of primary rat hepatocytes with well-differentiated Reuber H35 rat hepatoma cells. Seventeen hybrids were screened for bile acid synthesis using [26-14C]cholesterol. As [26-14C]cholesterol was converted to bile acid, 14CO2 was released. Using this assay, four hybrids (8B, 12C, 13C, and 13D) were identified which synthesized bile acid. These four hybrids also incorporated [14C]taurine into bile acid. Bile acids were identified by capillary gas chromatography/mass spectrometry, and their rates of synthesis were quantitated by isotope dilution. Reuber H35 cells synthesized little or no bile acid. However, hybrids 8B, 12C, 13C, and 13D synthesized chenodeoxycholic acid, alpha-muricholic acid, and cholic acid and secreted them into the media. The rates of synthesis of individual bile acids varied among these hybrids. For example, the relative percentage of cholic acid ranged from 11.1% (hybrid 8B) to 50.4% (hybrid 13C). The bile acids synthesized and secreted by the most active hybrid, 12C, were greater than 93% conjugated. In summary, hybrids were created that retain the capacity to synthesize, conjugate, and secrete three major rat bile acid species. Such hybrids are unique model systems that will allow the study of the biochemical and genetic regulation of bile acid synthesis.     

Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A simple, sensitive, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the determination of bile acids in human bile has been developed. The bile acids were extracted with a C(18) (octadecyl) reversed-phase column and identified and quantified by simultaneous monitoring of their parent and daughter ions, using the multiple reaction monitoring mode. Identification and quantification of conjugated bile acids in bile was achieved in 5 min. The detection limit was 1 ng, and the determination was linear for concentrations up to 100 ng. The percent recovery of standards made of single conjugated (glycine and taurine) bile acid or of mixture of glycine- or taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid averaged 71.73% to 95.92%. The percent recovery of the same standard bile acids was also determined by gas chromatography-mass spectrometry (GC-MS), using the selected ion monitoring mode, and averaged 66% to 96%. A biliary bile acid profile of human gallbladder bile was obtained by LC-MS/MS and GC-MS.The results showed a good correlation between the two techniques and no significant differences between the two methods were observed. The LC-MS/MS method was also used for the analysis of serum, urine, and fecal bile acids. In conclusion, LC-MS/MS is a simple, sensitive, and rapid technique for the analysis of conjugated bile acids in bile and other biological samples. - Perwaiz, S., B. Tuchweber, D. Mignault, T. Gilat, and I. M. Yousef. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J. Lipid Res. 2001. 42: 114;-119.     

Identification of ornithine and arginine conjugates of cholic acid by mass spectrometry.

Nalpha-Cholylornithine, -arginine, and -histidine were prepared according to a method previously employed for the chemical synthesis of the monoamino acid conjugates of bile acids. The products were shown to involve the alpha amino group of the dibasic amino acids by examination of the mass spectra of the original compounds, their lactams, their methyl esters and the methyl ester acetates. Only the methyl ester acetates gave detectable amounts of molecular ion. The free acids and the methyl esters of Nalpha-cholylornithine and -arginine gave identical lactams upon sublimation from the direct insertion probe. The synthetic Nalpha-cholylarginine was shown to yield a mass spectrum identical to that of an arginocholic acid recovered from the bile of an isolated perfused rat liver.     

胆汁酸代谢与肠道微生物

肠道微生物与胆汁酸代谢密切相关,肠道微生物参与了胆汁酸在肠道中的修饰过程;肠道微生物通过法尼醇受体影响胆汁酸的合成;肠道微生物通过调节胆汁酸的代谢影响机体健康,反过来胆汁酸也可以通过调节肠道微生物菌群的组成影响机体健康.肠道微生物与胆汁酸代谢间的稳态影响着机体健康,现对肠道微生物与胆汁酸代谢及其相互影响做一综述.     

Formylated bile acids: Improved synthesis,properties, and partial deformylation

Pure performylated bile acids are obtained in quantitative yield by anew formylation procedure. The procedure involves heating the bile acids in 90% formic acid containing catalytic amount of perchloric acid and then adding acetic anhydride slowly until effervescence occurs. Pure performylated bile acids are then isolated simply by diluting the reaction mixture with water. Contrary to what was believed by past investigations, the formyl groups on these compounds are quite stable to various reaction conditions. The stability and ready availability of these compounds make them more suitable candidates than their counterpart—bile acid acetates for use as starting material in various synthetic schemes, such as C-24 labeled bile acids, etc. The partial deformylation of these formates can be effected by using methanolic ammonia, sodium methoxide in methanol, or sodium hydroxide in aqueous acetone. The resulting 3-hydroxy formyl bile acids are obtained in high yield and are the best starting materials for the synthesis of bile acids with specific modification at 3-hydroxyl group, such as the synthesis of bile acid 3-monosulfates and 3-monoglucuronides.     

Bile acids reduce SR-BI expression in hepatocytes by a pathway involving FXR/RXR, SHP, and LRH-1

Hepatic SR-BI mediates uptake of circulating cholesterol into liver hepatocytes where a part of the cholesterol is metabolised to bile acids. In the hepatocytes, bile acids reduce their own synthesis by a negative feedback loop to prevent toxic high levels of bile acids. Bile acid-activated FXR/RXR represses expression of CYP7A1, the rate-limiting enzyme during bile acid synthesis, by inducing the expression of SHP, which inhibits LXR/RXR and LRH-1-transactivation of CYP7A1. The present paper presents data indicating that CDCA suppresses SR-BI expression by the same pathway. As previously reported, LRH-1 induces SR-BI promoter activity. Here we show that CDCA or over-expression of SHP inhibit this transactivation. No FXR-response element was identified in the bile acid-responsive region of the SR-BI promoter (-1200bp/-937bp). However, a binding site for LRH-1 was characterised and shown to specifically bind LRH-1. The present study shows that also the SR-BI-mediated supply of cholesterol, the substrate for bile acid synthesis, is feedback regulated by bile acids.     

Contraceptive steroids alter the steady-state kinetics of bile acids

Contraceptive steroids increase the ratio of cholic acid to chenodeoxycholic acid in bile. This alteration may contribute to the development of cholesterol gallstones. The objective of this study was to measure the effects of contraceptive steroids on bile acid kinetics and to relate them to changes in cholesterol metabolism. Steady-state kinetics of bile acids were measured in 15 healthy women, on and off contraceptive steroids. Cholic acid synthesis increased 30.3% (P less than 0.025) and its pool increased by 37.4% (P less than 0.025). Chenodeoxycholic acid synthesis decreased 6.4% (P = 0.08) and its pool decreased by 11.8% (P less than 0.05) during use of contraceptive steroids. The fractional turnover rates of both primary bile acids did not change. The changes in kinetics of the primary bile acids were related to alterations in biliary lipid and cholesterol metabolism, separately reported. (J. Lipid Res. 1987. 28: 828-839). During use of contraceptive steroids, total bile acid pool and total bile acid synthesis correlated directly with cholesterol synthesis, assayed in mononuclear leukocytes (r = 0.50 and r = 0.54, respectively) but not with the plasma clearance of chylomicron remnants, measured with retinyl palmitate. The data indicate that contraceptive steroids directly alter the hepatic synthesis of bile acids and suggest that newly synthesized cholesterol may be a preferred substrate for bile acid synthesis during use of contraceptive steroids.     

Bile acid synthesis: down-regulation by monohydroxy bile acids

The regulation of bile acid synthesis was studied in rabbits after interruption of the enterohepatic circulation by choledochoureteral anastomosis. Total daily bile acid output was 772 +/- 130 (SD) mumol/24 h, of which greater than 95% was glycocholic acid. Administration of deoxycholic or cholic acid or their conjugates (300-800 mumol) or gall-bladder bile failed to down-regulate endogenous bile acid synthesis. In contrast, chenodeoxycholic acid administration did down-regulate bile acid synthesis, but this effect was related to the formation and excretion of lithocholic acid. This observation was confirmed by the finding that i.v. infusion of 10-20 mumol of either lithocholic acid or 3 beta-hydroxy-5-cholenoic acid significantly reduced cholic acid synthesis. Thus monohydroxy bile acids, derived from either hepatic or intestinal sources, participate in the down-regulation of bile acid synthesis.     

Bile acid metabolism in early life: studies of amniotic fluid

Bile acid metabolism of the human fetus was examined in early gestation (weeks 13-19) and compared with the full-term fetus from the analysis of amniotic fluid collected from healthy pregnant women. Total individual bile acids were determined by gas-liquid chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. Additionally, bile acids were separated according to their mode of conjugation by lipophilic anion exchange chromatography. Qualitatively the bile acid profiles of amniotic fluid in early gestation were similar and markedly different from those of full-term fetuses. Chenodeoxycholic acid was the major bile acid identified in early gestation and concentrations exceeded those of cholic acid, but by full term this relationship was reversed. Over 50 bile acids were identified in the amniotic fluids, these included C-1, C-4, and C-6 hydroxylated species and reflected primary hepatic synthesis by the fetus. At full term, 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid was one of the major bile acids identified in amniotic fluid. The monohydroxy bile acids lithocholic and 3 beta-hydroxy-5-cholenoic acids were present in significant proportions during early gestation, but by full term these accounted for only a few percent of the total bile acids. Quantitatively the total bile acid concentration of amniotic fluid was less than 4 mumol/l. The majority of bile acids were found to be glyco-, tauro-, and sulfate-conjugates. The more hydrophobic bile acids tended to be preferentially sulfated. These data indicate that significant and major changes in bile acid metabolism take place between early and late gestation in the human fetus.     

Effect of mevinolin and glycyrrhizinic acid on cholesterol and bile acid metabolism in cultured rabbit hepatocytes]

A comparison of effects of two hypocholesterolemic drugs--mevinolin and glycyrrhizinic acid, on cholesterol and bile acid metabolism in cultured rabbit hepatocytes has been carried out. The following parameters have been determined: i) cholesterol synthesis from [2-14C]acetate; ii) bile acid production from newly synthesized and [4-14C]-labeled HDL2 cholesterol, and, iii) total cholesterol efflux into the incubation medium Mevinolin (0.5 microgram/ml) inhibited [2-14C] acetate incorporation into cholesterol by more than 90%. Conversely, glycyrrhizinic acid did not influence cholesterol synthesis even when used at high (100 micrograms/ml) concentrations but stimulated the conversion of endogenous (by 37%) and exogenous (by 18%) cholesterol into bile acids and increased, in addition, the proportion of bile acids in the total sterol pool released from hepatocytes into the incubation medium. At the same time, mevinolin used at 0.5 microgram/ml decreased the bile acid production by endogenous (by 27%) and exogenous (by 40%) cholesterol. The data obtained suggest that glycyrrhizinic acid exerts hypocholesterolemic action by stimulation of cholesterol conversion into bile acids without any effect on cholesterol synthesis. As for mevinolin, it has a cholesterol-suppressing effect via a mechanism of cholesterol synthesis inhibition only.