Effects of cholecystectomy on the kinetics of primary and secondary bile acids.

Removal of the gallbladder is thought to increase formation and pool size of secondary bile acids, mainly deoxycholic acid (DCA), by increased exposure of primary bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA]) to bacterial dehydroxylation in the intestine. We have tested this hypothesis by simultaneous determination of pool size and turnover of DCA, CA, and CDCA in nine women before and at various intervals after removal of a functioning gallbladder. An isotope dilution technique using marker bile acids labeled with stable isotopes (2H4-DCA, 13C-CA, 13C-CDCA) was used. After cholecystectomy, concentration and output of bile acids relative to bilirubin increased (P less than 0.02) in fasting duodenal bile and cholesterol saturation decreased by 27% (P less than 0.05) consistent with enhanced enterohepatic cycling of bile acids. Three months after removal of the gallbladder bile acid kinetics were in a new steady state: pool size and turnover of CDCA were unchanged. Synthesis of CA, the precursor of DCA, was diminished by 37% (P = 0.05), probably resulting from feedback inhibition by continuous transhepatic flux of bile acids. The fraction of CA transferred after 7 alpha-dehydroxylation to the DCA pool increased from 46 +/- 16 to 66 +/- 32% (P less than 0.05). However, this enhanced transfer did not lead to increased input or size of the DCA pool, because synthesis of the precursor CA had decreased.     

Immunosuppressive effects of endotoxins and bile acids in vivo in the rat

Cell-mediated immunity is impaired during cholestasis. The aim of this study was to evaluate in vivo the effects on this immune defect of high serum levels of endotoxin and bile acids. Heterotopic cardiac allotransplantations were performed in the DA/Lewis rat combination. Cholestasis, induced by ligation/section of the common bile duct, was responsible for a significant delay in the rejection time (16 ± 0.5 vs. 7.1 ± 0.4 days in controls, P <0.01). Elimination of Gram-negative intestinal bacteria from cholestatic rats by a vancocin/colimycin/tobramycin (VCT) mixture induced a significant reduction in endotoxin levels and a reduction in rejection times (9.5 ± 1.0 days, P <0.01) that remained, however, significantly longer than those of controls ( P <0.05). Oral administration of chenodeoxycholic acid in non-cholestatic rats significantly enhanced the serum concentration of total bile acids (60.6 ± 15.3 μmol L⁻¹ vs. 17.4 ± 1.9 μmol L⁻¹ in controls, P <0.01) and postponed allograft rejection (10.7 ± 0.6 days, P <0.01 vs. controls). These data suggest that increased endotoxin level and serum bile acid concentration may play a role in the immunosuppressive effect of cholestasis.     

Role of bile acid conjugation in hepatic transport of dihydroxy bile acids

The effect of conjugation and side chain length on dihydroxy bile acid unidirectional hepatic uptake and efflux was studied using the isolated perfused rat liver which was perfused prograde or retrograde in single pass fashion. Deoxycholic acid (DC) and its C23 (nor) derivative nor-DC, as well as the synthetically prepared taurine conjugate of DC, were administered at a constant dose of 1 mumol/min/kg (body weight), upon which a bolus tracer dose of labeled bile acid was superimposed. Analysis of radioactivity recovery in perfusate indicated that unidirectional uptake of all three bile acids was equally rapid, but that only nor-DC showed considerable and continuing efflux into the perfusate; this involved mostly the unchanged acid. Nor-DC was not amidated but was metabolized to mostly ester glucuronides and hydroxylated derivatives; the biotransformation products did not reflux and were secreted into bile; similarly, DC was amidated with taurine; its taurine conjugate did not efflux and was secreted into bile. When nor-DC-taurine was infused, it did not efflux and was secreted rapidly into bile. When the liver was perfused retrograde fashion to increase concentrations of bile acids pericentral cells, only nor-DC showed efflux, which again involved only the unchanged acid. All bile acids were partly 7 alpha-hydroxylated, the magnitude being greater during retrograde perfusion presumably because slower cellular transport exposed bile acid to hydroxylation enzymes for a longer period. It is concluded that bile acid conjugation, whether by esterification with CoA formation adn subsequent amidation or by esterification with glucuronate, restricts the movement of lipophilic dihydroxy bile acids to the hepatocyte and canalicular lumen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholic acid mediates negative feedback regulation of bile acid synthesis in mice

Cholesterol is converted into dozens of primary and secondary bile acids through pathways subject to negative feedback regulation mediated by the nuclear receptor farnesoid X receptor (FXR) and other effectors. Disruption of the sterol 12alpha-hydroxylase gene (Cyp8b1) in mice prevents the synthesis of cholate, a primary bile acid, and its metabolites. Feedback regulation of the rate-limiting biosynthetic enzyme cholesterol 7alpha-hydroxylase (CYP7A1) is lost in Cyp8b1(-/-) mice, causing expansion of the bile acid pool and alterations in cholesterol metabolism. Expression of other FXR target genes is unaltered in these mice. Cholate restores CYP7A1 regulation in vivo and in vitro. The results implicate cholate as an important negative regulator of bile acid synthesis and provide preliminary evidence for ligand-specific gene activation by a nuclear receptor.     

Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy.     

Impaired negative feedback suppression of bile acid synthesis in mice lacking betaKlotho

We have generated a line of mutant mouse that lacks betaKlotho, a protein that structurally resembles Klotho. The synthesis and excretion of bile acids were found to be dramatically elevated in these mutants, and the expression of 2 key bile acid synthase genes, cholesterol 7alpha-hydroxylase (Cyp7a1) and sterol 12alpha-hydroxylase (Cyp8b1), was strongly upregulated. Nuclear receptor pathways and the enterohepatic circulation, which regulates bile acid synthesis, seemed to be largely intact; however, bile acid-dependent induction of the small heterodimer partner (SHP) NR0B2, a common negative regulator of Cyp7a1 and Cyp8b1, was significantly attenuated. The expression of Cyp7a1 and Cyp8b1 is known to be repressed by dietary bile acids via both SHP-dependent and -independent regulations. Interestingly, the suppression of Cyp7a1 expression by dietary bile acids was impaired, whereas that of Cyp8b1 expression was not substantially altered in betaklotho mice. Therefore, betaKlotho may stand as a novel contributor to Cyp7a1-selective regulation. Additionally, betaKlotho-knockout mice exhibit resistance to gallstone formation, which suggests the potential future clinical relevance of the betaKlotho system.     

Enzymic measurement of primary bile acids and the primary bile acid ratio in serum with the IL-Multistat III Fluorescence Light-Scattering Centrifugal Analyzer

Enzymic fluorimetric methods are described for the determination of primary bile acids and of chenodeoxycholic acid (CDC) and cholic acid (C) in serum. Bile acids are extracted from 0.3 mL of serum in a simple 5-min step with use of Sep-Pak C cartridges. Total primary bile acids are measured by an equilibrium technique after reaction with beta-NAD in the presence of 7 alpha-hydroxysteroid dehydrogenase. Chenodeoxycholic acid (and its conjugates) is measured by a reaction-rate technique employing the same reaction as above but under different experimental conditions. A small contribution of cholic acid (and its conjugates) to the reaction rate is eliminated by simple calculations. Cholic acid is calculated by difference of the two determinations. In both assays NADH fluorescence is measured with the Multistat centrifugal analyzer. Absolute recovery of bile acids from serum was about 87%. Day-to-day standard deviations for CDC and C were 1.6 and 2.0 mumol/L at serum concentrations of 22.1 and 24.1 mumol/L respectively. Comparison data with a cholylglycine RIA procedure gave the following correlation coefficients (x = RIA, y = proposed method): r = 0.980 (RIA vs total primary bile acids), r = 0.918 (RIA vs CDC) and r = 0.989 (RIA vs C). The methods described appear more practical for use on a routine basis than methods in the literature for the calculation of the primary bile acid ratio.     

Erythrocyte membrane sialic acids in primary and secondary hypertension in man and rat

Abstract. Sialic acids of erythrocyte membranes (erythrocyte 'ghosts') and blood plasma were studied in patients with essential or chronic renal hypertension, and in spontaneously hypertensive rats (SHR). The total content of sialic acids in erythrocyte membranes (determined by thiobarbituric-acid assay) was around 100 μ mol/g protein in both the hypertensive patients and controls, there being no difference between the three groups. Similarly total sialic-acid content of plasma did not differ between the hypertensive patients and controls, being around 2 mmol/l. Although total membrane sialic acid was unchanged, the sialic-acid content of glycolipids extracted from erythrocyte membranes was 17% greater ( P < 0·001) in patients with essential hypertension than in renal hypertensive patients and controls (22·1 ± 0·5 μ mol/g protein v. 18·2 ± 0·8 and 18·9 ± 0·8, respectively). Sialic acid in plasma glycolipid did not differ between the patient groups. The animal study revealed no differences between total—or glycolipid—sialic-acid content in plasma and erythrocyte membrane in SHR and normotensive rats. The finding of an increase in the sialic-acid content of erythrocyte membrane glycolipid in essential hypertension is in agreement with recent studies demonstrating structural abnormalities in hydrophobic regions of erythrocyte membrane, and is considered a manifestation of membrane glycolipid alteration in primary hypertension.     

Serum cholesterol and bile acid in primary hepatoma.

The close relationship between serum levels of cholesterol and bile acid has been confirmed in 46 patients with primary hepatoma. Serum levels of cholesterol and bile acid are roughly correlated with serum alpha-fetoprotein concentration. Because the relationship between serum cholesterol and bile acid did not exist in common hepatocellular diseases, the results suggest a peculiar sterol metabolism occurring in human hepatoma.     

Role of bile acids in colon carcinogenesis

Bile acids (BAs) are cholesterol derivatives synthesized in the liver and then secreted into the intestine for lipid absorption. There are numerous scientific reports describing BAs, especially secondary BAs, as strong carcinogens or promoters of colon cancers. Firstly, BAs act as strong stimulators of colorectal cancer (CRC) initiation by damaging colonic epithelial cells, and inducing reactive oxygen species production, genomic destabilization, apoptosis resistance, and cancer stem cells-like formation. Consequently, BAs promote CRC progression via multiple mechanisms, including inhibiting apoptosis, enhancing cancer cell proliferation, invasion, and angiogenesis. There are diverse signals involved in the carcinogenesis mechanism of BAs, with a major role of epidermal growth factor receptor, and its down-stream signaling, involving mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt, and nuclear factor kappa-light-chain-enhancer of activated B cells. BAs regulate numerous genes including the human leukocyte antigen class I gene, p53, matrix metalloprotease, urokinase plasminogen activator receptor, Cyclin D1, cyclooxygenase-2, interleukin-8, and miRNAs of CRC cells, leading to CRC promotion. These evidence suggests that targeting BAs is an efficacious strategies for CRC prevention and treatment.     

Effects of cholic acid, 7 beta-hydroxy- and 12 beta-hydroxy-isocholic acid on bile flow, lipid secretion and bile acid synthesis in the rat

The effects of three epimeric trihydroxy-cholanoic acids, cholic acid (C), 7 beta-hydroxy-(7 beta) and 12 beta-hydroxy-(12 beta) isocholic acids on bile flow, lipid secretion, bile synthesis and bile micellar properties were studied in the rat with a bile fistula. The bile salts were infused intraduodenally starting 72 hours after cannulation when endogeneous bile salt synthesis had plateaued after the bile salt pool was drained. The bile salts were infused at two levels approximately 2 and 4 mumol min-1 kg-1. All three bile salts were absorbed and secreted almost quantitatively into the bile. Cholic acid was secreted in the conjugated form, 7 beta conjugated to approximately 60% and 12 beta completely in the unconjugated form. The bile salts did not undergo any significant biotransformations during the one passage from the intestine through the liver. Bile flow increased from the preinfusion level for all three bile salts infused in the order 7 beta greater than 12 beta greater than C. The bile flow increased linearly with bile salt secretion more for 7 beta than for C and 12 beta. Infusion of C increased the secretion into bile of phospholipid (PL) and cholesterol (CH) over the preinfusion values. Infusion of 7 beta as well as 12 beta resulted in a parallel decrease in the secretion of PL as well as CH compared to the preinfusion values. The infusion of C and 7 beta at the two levels used decreased the secretion of newly synthesized bile salt below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the 26-hydroxylase in the biosynthesis of bile acids in the normal state and in cerebrotendinous xanthomatosis. An in vivo study.

On the basis of different in vitro studies, we have previously suggested that the basic metabolic defect in the rare inherited disease cerebrotendinous xanthomatosis (CTX) is a lack of a hepatic mitochondrial C27-steroid 26-hydroxylase, involved in the normal biosynthesis of bile acids (1980. J. Clin. Invest. 65: 1418-1430; 1981. J. Lipid Res. 22: 191-200; 22: 632-640). In the present work, this hypothesis was tested in vivo. One patient with CTX and two control subjects received intravenously a mixture of [4-14C]7 alpha-hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha,26-dihydroxy-4-cholesten-3-one, steroids believed to be important precursors of chenodeoxycholic acid. The ratio between 14C and 3H in cholic acid and chenodeoxycholic acid isolated from bile of the CTX-patient was approximately 1/40 and 1/60 of those of the control subjects, respectively. Another patient with CTX and one control subject received a mixture of [4-14C]5 beta-cholestane-3 alpha,7 alpha-diol and [1,2-3H]5 beta-cholestane-3 alpha,7 alpha,26-triol, both possible precursors to chenodeoxycholic acid. In this case the 14C/3H ratio in cholic acid and chenodeoxycholic acid from the patient with CTX was 1/10 and 1/15, respectively, compared with that of the control subject. The most likely explanation for these findings is that very little of the 14C-precursors, i.e. without a 26-hydroxyl group, can be converted into cholic acid and chenodeoxycholic acid because of a defect of the 26-hydroxylase step. The results obtained are in accord with our previous findings in vitro. The results further underline the importance of the 26-hydroxylase pathway in the normal biosynthesis of cholic acid and chenodeoxycholic acid in man.     

Fasting serum bile acid levels in the primary hyperlipoproteinemias.

A newly developed radioimmunoassay specific for conjugates of cholic acid (CCA) was used to measure the fasting serum levels of these bile acids in 233 patients with primary hyperlipoproteinemia classified as type IIa, IIb, III, IV, or V hyperlipoproteinemia as well as in 80 healthy control subjects. Subjects with type IIa hyperlipoproteinemia had significantly lower levels of CCA (0.9 plus or minus 0.06 muM, mean plus or minus SE) than did healthy controls (0.50 plus or minus 0.08 muM). Patients with type IIb, III, IV, and V hyperlipoproteinemia had values similar to those of the control group. The abnormally low fasting value of CCA in type IIa patients is considered to reflect decreased secretion of bile acid into the intestine in such patients, who are known to have defective bile acid synthesis rates and decreased bile acid pool sizes.     

Metabolism of steroid and amino acid moieties of conjugated bile acids in man: II. Glycine-conjugated dihydroxy bile acids

Chenodeoxycholyl-2,4-(3)H-glycine-1-(14)C and deoxycholyl-2,4-(3)H-glycine-1-(14)C were synthesized and administered orally to 10 healthy subjects. Distribution of radioactivity among bile acids and specific activity of steroid and amino acid moieties were determined in bile samples. (3)H and (14)C were measured in feces. (14)C in breath was calculated from interval (14)CO(2) specific activity determinations.The daily fractional turnover of the glycine moiety of chenodeoxycholyl and deoxycholylglycines was more than three times that of the steroid moiety. Pool size of chenodeoxycholylglycine was about twice that of deoxycholylglycine, but similar fractional turnover rates of steroid and amino acid moieties suggested that intestinal absorption of the two conjugated bile acids was equally efficient (about 95%). The amount of unlabeled deoxycholic acid (newly formed by bacterial 7alpha-dehydroxylation) absorbed from the intestine approximated 30% of the cholic acid that was lost. (3)H radioactivity remained predominantly in administered bile acid implying that, normally, secondary bile acids derived from chenodeoxycholic acid are not appreciably absorbed from the intestine and that deoxycholic acid is not hydroxylated by the liver.Approximately 25% of administered (14)C was recovered in the breath in the first 24 hr and less than 8% in the feces in 8 days; (14)CO(2) excretion correlated highly with fractional turnover of the glycine moiety. (3)H appeared predominantly in feces, and the rate of excretion correlated highly with the fractional turnover of the steroid moiety of bile acids. From the results in this paper plus previous measurements on the metabolism of cholylglycine, we calculated that about 6 mmoles/day of glycine is used for bile acid conjugation in health.     

Inappropriate hepatic cholesterol synthesis expands the cellular pool of sterol available for recruitment by bile acids in the rat.

These studies test the hypothesis that a major determinant of excessive biliary cholesterol secretion is a level of hepatic sterol synthesis that is inappropriately high relative to the needs of the liver cell for preserving cholesterol balance. Biliary cholesterol secretion was measured in vivo in two models after loading the hepatocyte with sterol by two different mechanisms. In the first model, cholesterol was delivered physiologically to the liver in chylomicron remnants. This resulted in a sixfold increase in cholesteryl ester content and marked suppression of cholesterol synthesis, but biliary cholesterol secretion remained essentially constant. In the second model, 3-hydroxy-3-methyl-glutaryl CoA reductase levels in the liver were markedly increased by chronic mevinolin (lovastatin) administration. Withdrawal of the inhibitor resulted in a sudden fivefold increase in the rate of sterol synthesis in the liver of the experimental animals that was inappropriately high for cellular needs. This excessive synthesis, in turn, was accompanied by a fivefold increase in the cholesteryl ester content, enrichment of microsomal membranes with cholesterol and, most importantly, by a threefold increase in the rate of biliary sterol secretion. As the rate of sterol synthesis gradually returned to normal over 48 h, the cholesterol ester content, the lipid composition of the microsomal membranes, and rate of cholesterol secretion into bile also returned to baseline values. These results further support the concept of functional compartmentalization of cholesterol in the hepatocyte. Derangements that cause an inappropriately high rate of sterol synthesis in the endoplasmic reticulum may lead to an expansion of that pool of cholesterol that is recruitable by bile acids and, hence, to greater situation of the bile.     

The preparation of 125I-labelled bile acid ligands for use in the radioimmunoassay of bile acids.

A general method for the preparation of 125I-labelled bile acid-histamine or 125I-labelled bile acid-tyramine conjugates is presented. The method is simple, quick and produces ligands in good yield (30%). The characteristics of a radioimmunoassay for conjugated chenodeoxycholic acid, based on an 125I-labelled ligand prepared by the method, are also described. The assay produced values for fasting serum concentrations of conjugated chenodeoxycholic acid that agree well with previous data.     

Cholestasis induced by sulphated glycolithocholic acid in the rat: protection by endogenous bile acids

Sulphated glycolithocholic acid (SGLC) causes cholestasis in experimental animals, despite its sulphated form. In the present study, the cholestatic potency and the pharmacokinetics of SGLC were investigated in rats under two conditions: (a) in the presence of an intact circulating bile acid pool and (b) after exhaustion of the bile acid pool by 24 h of bile diversion. Intravenous administration of SGLC (8 mumol/100 g body weight) to rats with an intact bile acid pool did not cause cholestasis. However, biliary phospholipid and cholesterol concentrations were reduced by 40% and 29% respectively during the first hour after administration. When the same dose of the bile acid was injected in rats with a 24 h biliary drainage, a complete cessation of bile production was observed within 1 h. Twelve hours after the onset of cholestasis, bile production gradually increased again, showed a marked overshoot, and reached control levels after 3 days. In the recovery phase, biliary phospholipid and cholesterol concentrations were greatly reduced. The absence of endogenous bile acids did not change the hepatic clearance rate of a tracer dose of radiolabelled SGLC, but markedly decreased its biliary excretion rate. It was concluded that the hepatotoxic effect of SGLC is much more pronounced in rats with an exhausted bile acid pool, possibly due to a slower biliary excretion of the toxic compound. This phenomenon may have clinical implications for patients with a contracted bile acid pool.     

Growth hormone and bile acid synthesis. Key role for the activity of hepatic microsomal cholesterol 7alpha-hydroxylase in the rat.

Growth hormone (GH) has an important role in the regulation of hepatic LDL receptor expression and plasma lipoprotein levels. This investigation was undertaken to characterize the effects of GH on hepatic cholesterol and bile acid metabolism in the rat. In hypophysectomized (Hx) rats, the activities of the rate-limiting enzymes in cholesterol and bile acid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and cholesterol 7alpha-hydroxylase (C7alphaOH), were reduced by 71 and 64%, respectively. HMG CoA reductase mRNA levels were reduced by 37%, whereas C7alphaOH mRNA was increased by 81%. LDL receptor expression was reduced by 18% in Hx rats, without any change in the LDL receptor mRNA levels. Although the normal diurnal variation of C7alphaOH activity was preserved in Hx rats, the activity of C7alphaOH was much reduced both at midday and midnight. Total hepatic cholesterol was increased by 14% in Hx animals whereas microsomal cholesterol was unchanged. The rate of cholesterol esterification was enhanced (by 38%) in liver microsomes from Hx rats. Stepwise hormonal substitution of Hx rats showed that GH, but not thyroid hormone or cortisone, was essential to normalize the enzymatic activity of C7alphaOH. GH also normalized the altered plasma lipoprotein pattern in Hx rats, and increased the fecal output of bile acids. The latter effect was particularly evident when GH was combined with cortisone and thyroid hormone. Also in normal rats, GH stimulated C7alphaOH activity. In conclusion, GH has an essential role to maintain a normal enzymatic activity of C7alphaOH, and this, at least in part, explains the effects of GH on hepatic cholesterol metabolism. GH is also of critical importance to normalize the altered plasma lipoprotein pattern in Hx rats.