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The CYP27A gene encodes a mitochondrial cytochrome P450 enzyme, sterol 27-hydroxylase, that is expressed in many different tissues and plays an important role in cholesterol and bile acid metabolism. In humans, CYP27A deficiency leads to cerebrotendinous xanthomatosis. To gain insight into the roles of CYP27A in the regulation of cholesterol and bile acid metabolism, cyp27A gene knockout heterozygous, homozygous, and wild-type littermate mice were studied. In contrast to homozygotes, heterozygotes had increased body weight and were mildly hypercholesterolemic, with increased numbers of lipoprotein particles in the low density lipoprotein size range. Cyp7A expression was not increased in heterozygotes but was in homozygotes, suggesting that parts of the homozygous phenotype are secondary to increased cyp7A expression and activity. Homozygotes exhibited pronounced hepatomegaly and dysregulation in hepatic cholesterol, bile acid, and fatty acid metabolism. Hepatic cholesterol synthesis and synthesis of bile acid intermediates were increased; however, side chain cleavage was impaired, leading to decreased bile salt concentrations in gallbladder bile. Expression of Na-taurocholate cotransporting polypeptide, the major sinusoidal bile salt transporter, was increased, and that of bile salt export pump, the major canalicular bile salt transporter, was decreased. Gender played a modifying role in the homozygous response to cyp27A deficiency, with females being generally more severely affected. Thus, both cyp27A genotype and gender affected the regulation of hepatic bile acid, cholesterol, and fatty acid metabolism.

Dietary cholesterol regulation of cholesterol 7alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme in the classical pathway of bile acid synthesis, has been implicated in plasma cholesterol responsiveness. In the current study, the effects of 0.0% and 0.5% cholesterol diets were examined in Cyp7a1 knockout (KO), heterozygous Cyp7a1 KO (Het), and human Cyp7a1 transgenic mice on the mouse Cyp7a1 KO background (Tg+KO). We confirmed previous findings that dietary cholesterol increased mouse Cyp7a1 activity in Het mice but decreased human Cyp7a1 activity in Tg+KO mice. However, in both Het and Tg+KO mice, dietary cholesterol increased bile acid pool size (36% and 72%, respectively) and fecal bile acid excretion (2.2- and 3.6-fold, respectively). The expression of cholesterol 27-hydroxylase (Cyp27), the major enzyme of the alternative pathway of bile acid synthesis, was not significantly different in cholesterol-fed KO, Het, or Tg+KO mice. Furthermore, dietary cholesterol had comparable effects on total plasma cholesterol and non-high-density lipoprotein cholesterol in KO, Het, and Tg+KO mice. Thus, in Tg+KO mice, dietary cholesterol regulates bile acid pool size, fecal bile acid excretion, and plasma cholesterol independently of Cyp7a1 activity. These results challenge the notion that dietary cholesterol regulation of Cyp7a1 is a major determinant of plasma cholesterol responsiveness.

Plasma 7alpha-hydroxy-4-cholesten-3-one has been used as an index of hepatic bile acid synthesis. The aim of the current study was to ascertain whether the level of this oxysterol reflects hepatic cholesterol 7alpha-hydroxylase activity when plasma cholesterol concentrations are markedly changed. In addition, the relationship of hepatic sterol 27-hydroxylase activity with plasma concentrations of 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid was studied. We used New Zealand white rabbits fed 2% cholesterol for 5 or 10 days and/or constructed bile fistula. Feeding cholesterol markedly increased and bile drainage reduced plasma cholesterol concentrations. Initially, in these models there was no correlation between plasma 7alpha-hydroxy-4-cholesten-3-one concentrations and hepatic cholesterol 7alpha-hydroxylase activities (r = -0.24, n = 10). Cholesterol feeding was associated with downregulated 7alpha-hydroxylase activities, while plasma 7alpha-hydroxy-4-cholesten-3-one concentrations were elevated in the presence of increased plasma cholesterol levels. However, this discrepancy was overcome and significant correlation was observed (r = 0.73, P <.05, n = 10) by expressing 7alpha-hydroxy-4-cholesten-3-one levels relative to cholesterol. In contrast, hepatic sterol 27-hydroxylase activities were not significantly correlated with plasma absolute (r = 0.23, difference not significant [NS], n = 10) nor cholesterol-related levels of 27-hydroxycholesterol (r = -0.13, NS, n = 10), or 3beta-hydroxy-5-cholestenoic acid concentrations (r = 0.30, NS, n = 10). In conclusion, plasma 7alpha-hydroxy-4-cholesten-3-one concentrations reflected hepatic cholesterol 7alpha-hydroxylase activities when the sterol levels were adjusted to plasma cholesterol concentrations in rabbits with hypercholesterolemia. The results suggest that plasma 7alpha-hydroxy-4-cholesten-3-one relative to cholesterol is a better marker for hepatic cholesterol 7alpha-hydroxylase activity than the absolute concentration when hypercholesterolemia is present. In contrast, 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid levels in plasma did not reflect hepatic sterol 27-hydroxylase activities even if the levels were adjusted to plasma cholesterol concentrations.

Cholesterol 7alpha-hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1, encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile. Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.

We investigated the roles of hydrophobic deoxycholic acid (DCA) and hydrophilic ursocholic acid (UCA) in the regulation of the orphan nuclear farnesoid X receptor (FXR) in vivo. Rabbits with bile fistula drainage (removal of the endogenous bile acid pool), rabbits with bile fistula drainage and replacement with either DCA or UCA, and intact rabbits fed 0.5% cholic acid (CA) (enlarged endogenous bile acid pool) were studied. After bile fistula drainage, cholesterol 7alpha-hydroxylase (CYP7A1) mRNA and activity levels increased, FXR-mediated transcription was decreased, and FXR mRNA and nuclear protein levels declined. Replacing the enterohepatic bile acid pool with DCA restored FXR mRNA and nuclear protein levels and activated FXR-mediated transcription as evidenced by the increased expression of its target genes, SHP and BSEP, and decreased CYP7A1 mRNA level and activity. Replacing the bile acid pool with UCA also restored FXR mRNA and nuclear protein levels but did not activate FXR-mediated transcription, because the SHP mRNA level and CYP7A1 mRNA level and activity were unchanged. Feeding CA to intact rabbits expanded the bile acid pool enriched with the FXR high affinity ligand, DCA. FXR-mediated transcription became activated as shown by increased SHP and BSEP mRNA levels and decreased CYP7A1 mRNA level and activity but did not change FXR mRNA or nuclear protein levels. Thus, both hydrophobic and hydrophilic bile acids are effective in maintaining FXR mRNA and nuclear protein levels. However, the activating ligand (DCA) in the enterohepatic flux is necessary for FXR-mediated transcriptional regulation, which leads to down-regulation of CYP7A1.

Ileal reclamation of bile salts is mediated in large part by an apical sodium-dependent bile acid transporter (ASBT) located in the terminal ileum. The following studies were performed to elucidate the adaptive response of ASBT to intestinal resection. Two separate series of intestinal resections were performed: 1) limited (25%) ileal and 2) massive (70%) intestinal resection. The boundaries of the resections were varied to examine differences in compensation when variable amounts of endogenous transporter activity were resected. Previously demonstrated supraphysiologic expression of ASBT, which was seen after proximal ileal resection, led to a contraction in the bile acid pool size and a paradoxical reduction in bile acid (cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase) and cholesterol (hydroxymethylglutaryl coenzyme A reductase) biosynthetic enzyme activities. Massive intestinal resection resulted in ileal hypertrophy and an apparently maladaptive specific down-regulation in ASBT protein expression. In this model bile acid pool size correlated with the amount of residual ASBT-expressing terminal ileum. Cholesterol and bile acid biosynthetic enzyme activities were inversely related to bile acid pool size. Adaptive changes in ASBT expression and alterations in bile acid and cholesterol homeostasis are dependent on the type and location of intestinal resection.

We investigated the role of the orphan nuclear receptor farnesoid X receptor (FXR) in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1), using an in vivo rabbit model, in which the bile acid pool, which includes high affinity ligands for FXR, was eliminated. After 7 days of bile drainage, the enterohepatic bile acid pool, in both New Zealand White and Watanabe heritable hyperlipidemic rabbits, was depleted. CYP7A1 activity and mRNA levels increased while FXR was deactivated as indicated by reduced FXR protein and changes in the expression of target genes that served as surrogate markers of FXR activation in the liver and ileum, respectively. Hepatic bile salt export pump mRNA levels and ileal bile acid-binding protein decreased while sterol 12alpha-hydroxylase and sodium/taurocholate cotransporting polypeptide mRNA levels increased in the liver. In addition, hepatic FXR mRNA levels decreased significantly. The data, taken together, indicate that FXR was deactivated when the bile acid pool was depleted such that CYP7A1 was upregulated. Further, lack of the high affinity ligand supply was associated with downregulation of hepatic FXR mRNA levels.

The accumulation of various 25-hydroxylated C(27)-bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27(-/-) mice do not show CTX-related abnormalities. Microsomal activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6beta-hydroxylase, a marker enzyme for CYP3A, in Cyp27(-/-) mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases and 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6beta-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dose-dependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25- and 26-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27(-/-) mice, CYP3A activity was not up-regulated despite marked accumulation of 5beta-cholestane-3alpha,7alpha,12alpha-triol in CTX.

To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 +/- 55 to 444 +/- 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 +/- 77 vs. 276 +/- 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 +/- 6 to 136 +/- 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 +/- 14 vs. 99 +/- 19 units/mg) after cholesterol feeding. Cholesterol 7alpha-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol. Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7alpha-hydroxylase was not inhibited.

We compared hepatic cholesterol metabolism in apolipoprotein (apo) E-knockout (KO) mice with their wild-type counterparts. We also investigated the effects of treatment with phytosterols or probucol on the activity of hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (cholesterol synthesis), cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase (bile acid synthesis), and low-density lipoprotein (LDL) receptor function in this animal model of atherogenesis. These findings were then related to treatment-induced changes in plasma, hepatic, and fecal sterol concentrations. Mouse liver membranes have binding sites similar to LDL receptors; the receptor-mediated binding represents 80% of total binding and is LDL concentration-dependent. These binding sites have higher affinity for apo E-containing particles than apo B only-containing particles. Deletion of apo E gene was associated with several-fold increases in plasma cholesterol levels, 1.5-fold increase in hepatic cholesterol concentrations, 50% decrease in HMG-CoA reductase activity, 30% increase in cholesterol 7 alpha-hydroxylase and 25% decrease in LDL receptor function. Treatment of apo E-KO mice with either probucol or phytosterols significantly reduced plasma cholesterol levels. Phytosterols significantly increased the activity of hepatic HMG-CoA reductase, and probucol significantly increased cholesterol 7 alpha-hydroxylase activity. Neither treatment significantly altered hepatic LDL receptor function. Phytosterols, but not probucol, significantly increased fecal sterol excretion and decreased hepatic cholesterol concentrations. Plasma cholesterol lowering effects of phytosterols and probucol are due to different mechanisms: stimulation of cholesterol catabolism via increased bile acid synthesis by probucol and decreased cholesterol absorption by phytosterols. In the absence of apo E, hepatic LDL receptors could not be upregulated and did not contribute to the cholesterol lowering effects of either agent., (Copyright 2001 by W.B. Saunders Company)

Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1alpha (encoded by TCF1). Tcf1-/- mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1-/- mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1-/- liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1-/- mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1alpha) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1-/- mice. Increased plasma cholesterol of Tcf1-/- mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27(-/-) and 7 Cyp27(+/+) mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5beta-cholestane-3alpha,7alpha,12alpha,27-tetrol were virtually absent in both Cyp27(-/-) mice and CTX patients. In Cyp27(-/-) mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7alpha-hydroxycholesterol, 7alpha-hydroxy-4-cholesten-3-one, 7alpha,12alpha-dihydroxy-4-cholesten-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol), 25-hydroxylated bile alcohols (5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, 5beta-cholestane-3alpha,7alpha,12alpha,23R,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27(+/+) mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16;-86% of CTX) than in female Cyp27(-/-) mice (7-30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27(-/-) mice and were considerably lower (less than 14%) than those in CTX patients.These results suggest that 1) in Cyp27(-/-) mice, especially in females, classic bile acid biosynthesis via 7alpha-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27(-/-) mice than in CTX patients.

Background & Aims: The mechanism for abnormal hepatic bile acid transport was investigated in an 18-month-old Amish boy who presented with pruritus, poor growth, and severe bleeding episodes. Serum bilirubin, gamma-glutamyltranspeptidase, and cholesterol levels were normal, but prothrombin time and partial thromboplastin time were prolonged and bone alkaline phosphatase level was elevated., Methods and Results: Cholic acid plus chenodeoxycholic acid levels measured by capillary gas-chromatography were 32 times higher than control in serum (34.7 vs. 1.1+/-0.4 microg/dL) but were not detected in liver and were reduced in gallbladder bile. Treatment with ursodiol, a more hydrophilic bile acid, improved pruritus, produced 37% weight gain, and after 2 years reduced serum primary bile acid concentrations about 85%, while accounting for 71% of serum and 24% of biliary bile acid conjugates. On ursodiol therapy, hepatic bile acid synthesis was enhanced 2-fold compared with controls, and microscopy revealed chronic hepatitis without cholestasis. Three younger sisters with elevated serum bile acids responded positively to ursodiol. Microsatellite markers for the FIC1 (gene for Byler's disease) region in these 4 children were inconsistent with linkage to FIC1., Conclusions: Conjugated cholic acid and chenodeoxycholic acid were synthesized in the liver and secreted into bile but could not reenter the liver from portal blood and accumulated in serum. In contrast, unconjugated ursodiol entered the liver and was conjugated and secreted into bile. Thus, the enterohepatic circulation of all conjugated bile acids was interrupted at the hepatic sinusoidal basolateral membrane. Unconjugated ursodiol bypassed the hepatic uptake block to enlarge the biliary and intestinal bile acid pools. A mutation in FIC1 recognized among the Amish and linkage of the disorder to FIC1 were excluded.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7alpha-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 +/- 19 mg/dl) and 31-times (986 +/- 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 +/- 17 mg vs. 61 +/- 18 mg) in rats but doubled (254 +/- 46 to 533 +/- 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7alpha-hydroxylase activity rose 33% (24 +/- 2.4 vs. 18 +/- 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7alpha-hydroxylase in rabbits.

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5beta-cholestane-3alpha,7alpha-diol, and 5beta-cholestane-3alpha,7alpha,12alpha-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7alpha-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7alpha-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7alpha-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.

The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3beta-hydroxysterol Delta(7)-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Delta(7)-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7alpha-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7alpha-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3beta-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS.

We investigated the effect of increasing dietary cholesterol on bile acid pool sizes and the regulation of the two bile acid synthetic pathways (classic, via cholesterol 7alpha-hydroxylase, and alternative, via sterol 27-hydroxylase) in New Zealand white rabbits fed 3 g cholesterol/per day for up to 15 days. Feeding cholesterol for one day increased hepatic cholesterol 75% and cholesterol 7alpha-hydroxylase activity 1.6 times without significant change of bile acid pool size or sterol 27-hydroxylase activity. After three days of cholesterol feeding, the bile acid pool size increased 83% (P < 0.01), and further feeding produced 10%-20% increments, whereas cholesterol 7alpha-hydroxylase activity declined progressively to 60% below baseline. In contrast, sterol 27-hydroxylase activity rose 58% after three days of cholesterol feeding and remained elevated with continued intake. Bile drainage depleted the bile acid pool and stimulated downregulated cholesterol 7alpha-hydroxylase activity but did not affect sterol 27-hydroxylase activity. Thus, increasing hepatic cholesterol does not directly inhibit cholesterol 7alpha-hydroxylase and initially favors enzyme induction, whereas increased bile acid pool is the most powerful inhibitor of cholesterol 7alpha-hydroxylase. Sterol 27-hydroxylase is insensitive to the bile acid flux but is upregulated by increasing hepatic cholesterol.

The effect of cholesterol feeding (3 g/day) on bile acid synthesis was examined in 10 New Zealand white rabbits (NZW), 8 Watanabe heterozygous and 10 homozygous rabbits with partial and complete deficiencies of LDL receptors. After 10 days of cholesterol feeding, bile fistulas were constructed and bile acid pool sizes were measured. Cholesterol feeding increased plasma and hepatic cholesterol levels in all rabbit groups. Baseline bile acid pool sizes were smaller (P < 0.01) in heterozygotes (139 +/- 3 mg) and homozygotes (124 +/- 30 mg) than NZW rabbits (254 +/- 44 mg). After feeding cholesterol, bile acid pool sizes doubled with increased cholic acid synthesis in NZW and, to a lesser extent, in Watanabe heterozygous rabbits but not in homozygotes. Baseline cholesterol 7alpha-hydroxylase activity in NZW and heterozygotes declined 69% and 53% (P < 0.001), respectively, after cholesterol feeding. Sterol 27-hydroxylase activity reflecting alternative bile acid synthesis increased 66% (P < 0.01) in NZW and 37% in Watanabe heterozygotes but not in homozygotes after feeding cholesterol. Bile fistula drainage stimulated cholesterol 7alpha-hydroxylase activity but not sterol 27-hydroxylase activity in all three rabbit groups. These results demonstrated that dietary cholesterol increased hepatic sterol 27-hydroxylase activity and alternative bile acid synthesis to expand the bile acid pool and inhibited cholesterol 7alpha-hydroxylase in NZW and in Watanabe heterozygous rabbits but not in homozygotes with absent hepatic LDL receptor function. Thus, in rabbits, sterol 27-hydroxylase is up-regulated by the increased hepatic cholesterol that enters the liver via LDL receptors whereas cholesterol 7alpha-hydroxylase is controlled by the circulating hepatic bile acid flux.

Background & Aims: Cholesterol feeding unexpectedly inhibits cholesterol 7 alpha-hydroxylase in rabbits. The aim of this study was to explore the mechanism., Methods: Twenty male New Zealand white rabbits were fed regular chow with and without 2% cholesterol for 10 days followed by 7 days of bile drainage. The activities of hepatic cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase that control bile acid synthesis in classic and alternative pathways were related to the size and composition of bile acid pool., Results: After feeding cholesterol, plasma and hepatic cholesterol concentrations increased, the bile acid pool doubled (from 254 +/- 44 to 533 +/- 51 mg; P < 0.001), cholesterol 7 alpha-hydroxylase activity decreased 68% (P < 0.01), but sterol 27-hydroxylase activity increased 66% (P < 0.05) with increased cholic acid synthesis (P < 0.01). Bile drainage in the cholesterol-fed rabbits depleted the bile acid pool and stimulated down-regulated cholesterol 7 alpha-hydroxylase activity 11.4-fold (P < 0.001), although hepatic cholesterol remained elevated. Hepatic sterol 27-hydroxylase activity was unaffected., Conclusions: Feeding cholesterol increased hepatic cholesterol and stimulated sterol 27-hydroxylase and alternative bile acid synthesis, which expanded the bile acid pool and inhibited cholesterol 7 alpha-hydroxylase in rabbits. In distinction, hepatic sterol 27-hydroxylase was insensitive to changes in the bile acid pool.

N-nitrosoamides of 7 beta-hydroxylated bile acid conjugates, particularly of the ursodeoxycholic acid family have been synthesized. The products and synthetic intermediates were fully characterized by the results of high-resolution 1H NMR, FT-IR, FABMS and ESI-MS studies. The compounds, N-nitrosoglycoursodeoxycholic acid (NOGUDCA), N-nitrosoglycoursocholic acid (NOGUCA) and N-nitrosoglycodeoxycholic acid (NOGDCA) decomposed between pH 6 and 9 in aqueous buffer solutions, indicating a t1/2 of 5-7 h while N-nitrosotauroursodeoxycholic acid (NOTUDCA) indicated a much longer t1/2 of 15-17 h. These results suggest that the compounds are relatively stable and may enter the enterohepatic circulation. Their decomposition is similar to that of other N-nitrosamides, which generate alkylating agents and thereby act as DNA mutagens.

We examined the effects of feeding deoxycholic acid (1% and 0.4% of diet), alone and in combination with ursodeoxycholic acid, on serum and biliary bile acid concentrations, hepatic morphology, and the activities and steady-state messenger RNA (mRNA) levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in the rat. Feeding 1% deoxycholic acid increased serum bile acid concentrations (cholestasis), produced portal triad inflammation, bile duct proliferation, and severe hepatocyte necrosis with nuclear pleomorphism. Hepatic damage was prevented when ursodeoxycholic acid (1%) was combined with the deoxycholic acid (1%), or when deoxycholic acid intake was reduced to 0.4%. HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities were markedly inhibited (-56% and -55%, respectively) with either 1% or 0.4% deoxycholic acid. Ursodeoxycholic acid alone produced an insignificant decline in HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities, and when combined with 1% deoxycholic acid did not lessen the inhibitory effect of the latter. Steady-state mRNA levels increased 20-fold for HMG-CoA reductase and 53-fold for cholesterol 7 alpha-hydroxylase in rats fed 1% deoxycholic acid. In contrast, 0.4% deoxycholic acid decreased HMG-CoA reductase mRNA levels 76%, and cholesterol 7 alpha-hydroxylase mRNA levels 82%. Ursodeoxycholic acid alone did not affect HMG-CoA reductase or cholesterol 7 alpha-hydroxylase steady-state mRNA levels. Steady-state mRNA levels and activities of sterol 27-hydroxylase, a key enzyme in the alternative acidic pathway of bile acid synthesis, did not change with either high or low doses of deoxycholic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

The Smith-Lemli-Opitz syndrome is a recessive inherited disorder characterized by neurologic developmental defects and dysmorphic features in many organs. Recently, abnormal cholesterol biosynthesis with impaired conversion of 7-dehydrocholesterol to cholesterol has been discovered in homozygotes. To reproduce the biochemical abnormality, BM 15.766, a competitive inhibitor of 7-dehydrocholesterol-delta 7-reductase, the enzyme that catalyzes the conversion of 7-dehydrocholesterol into cholesterol was fed by gavage to rats. After 14 d, plasma cholesterol concentrations declined from 48 mg/dl to 16 mg/dl and 7-dehydro-cholesterol levels rose from trace to 17 mg/dl. Hepatocytes surrounding the central vein developed balloon necrosis. Stimulating cholesterol synthesis with cholestyramine followed by BM 15.766 produced an additional 40% decline (P < 0.05) in plasma cholesterol and 34% increase in 7-dehydrocholesterol levels compared to the inhibitor alone. Adding 2% cholesterol to the diet during the second week of BM 15.766 treatment increased plasma cholesterol threefold and decreased 7-dehydrocholesterol concentrations 55%. Hepatic 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase activity increased 73% with a 3.9-fold rise in mRNA levels but cholesterol 7 alpha-hydroxylase activity decreased slightly though mRNA levels increased 1.4 times with BM 15.766 treatment. These results demonstrate that BM 15.766 is a potent inhibitor of 7-dehydrocholesterol-delta 7-reductase. The model reproduces abnormal cholesterol biosynthesis as seen in the Smith-Lemli-Opitz syndrome and is useful to test different treatment strategies. Stimulating early steps of cholesterol synthesis worsens the biochemical abnormalities while feeding cholesterol inhibits abnormal synthesis, improves the biochemical abnormalities and prevents liver damage.

We investigated the effects of intestinal bile acid flux, orientation of the 7-hydroxy group, and administration of lovastatin on the regulation of intestinal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity in the rat ileum. HMG-CoA reductase activities in villous and crypt cells from the ileal mucosa were similar, and the study was performed on whole mucosa that contained both cell types. Taurocholate feeding decreased ileal reductase activity 48%, whereas tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, had no effect. Feeding lovastatin (inhibitor of HMG-CoA reductase) stimulated total ileal HMG-CoA reductase activity threefold in washed microsomes, which were dissociated from the inhibitor. However, the proportion of active enzyme in the ileum of lovastatin-fed rats was 50% lower than in controls, whereas there was no change in the percentage of expressed enzyme with bile acid treatments. Interruption of the enterohepatic circulation (bile fistula) increased HMG-CoA reductase activity in the ileum 73%. Duodenal infusion of taurocholate to bile-fistula rats significantly decreased microsomal HMG-CoA reductase activity in the ileal mucosa. In contrast, infusion of the 7 beta-hydroxy epimer tauroursocholate failed to inhibit the derepressed HMG-CoA reductase activity in the ileum of bile-fistula rats. The inhibition of intestinal HMG-CoA reductase activity by taurocholate occurred without accumulation of mucosal cholesterol. Furthermore, the stimulation of total ileal HMG-CoA reductase activity by lovastatin treatment was observed without a decrease in mucosal cholesterol. In summary, the regulation of ileal HMG-CoA reductase activity by the intestinal luminal flux of bile acids is dependent on the orientation of the hydroxyl groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the relationship between plasma cholestanol (5 alpha-dihydrocholesterol) concentrations and the activity and mRNA levels of cholesterol 7 alpha-hydroxylase, the rate-controlling enzyme for bile acid synthesis, in three female sitosterolemic homozygotes. In this lipid storage disease, large amounts of plant sterols and cholestanol accumulate because of hyperabsorption and endogenous synthesis, respectively. Plasma cholestanol concentrations were 14 times greater in the three sitosterolemic homozygotes than the mean for five control subjects. To investigate the cholestanol biosynthetic pathway, tracer doses of two putative precursors, [1,2-3H]4-cholesten-3-one and [4-14C]7 alpha-hydroxycholesterol were injected intravenously into a homozygote, and radioactivity was sought in cholestanol, bile acids, cholesterol, and sitosterol fractions isolated from plasma and bile. Tritium was concentrated only in cholestanol; neither cholesterol, sitosterol nor bile acids were derived from [1,2-3H]4-cholesten-3-one. In contrast, bile acids were labeled exclusively with 14C from [4-14C]7 alpha-hydroxycholesterol; no 14C radioactivity was detected in cholestanol. Mathematical analysis of specific activity versus time curves for [3H]cholestanol revealed very slow decay, large exchangeable pools, and enhanced synthesis in the sitosterolemic homozygote. Measurements of cholesterol 7 alpha-hydroxylase activity were 39% lower in whole liver microsomes from three sitosterolemic homozygotes that contained 19% plant sterols as compared to the mean value for six control microsomal specimens that contained 0.1% plant sterols. Removal of the excess plant sterols from the microsomes, in vitro, normalized microsomal cholesterol 7 alpha-hydroxylase activity in the homozygotes but did not affect enzyme activity in the controls. Equal amounts of cholesterol 7 alpha-hydroxylase mRNA were detected in the livers of both control and sitosterolemic subjects. Bile acid malabsorption after ileal bypass surgery stimulated cholesterol 7 alpha-hydroxylase activity 78% in sitosterolemic whole liver microsomes and reduced plasma cholesterol, sitosterol, and cholestanol levels 61%, 55% and 91%, respectively, producing a pronounced decrease in the cholestanol/cholesterol ratio without changing the sitosterol/cholesterol ratio. These results demonstrate that increased cholestanol is synthesized from 4-cholesten-3-one and not 7 alpha-hydroxycholesterol in sitosterolemia. Enhanced pools and plasma concentrations are related inversely to hepatic cholesterol 7 alpha-hydroxylase activity. Competitive inhibition of cholesterol 7 alpha-hydroxylase by the large microsomal plant sterol pool diverts cholesterol into cholestanol. Alternatively, stimulating cholesterol 7 alpha-hydroxylase activity after ileal bypass surgery markedly diminished plasma cholestanol levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Background: To explore the sexual difference in the feedback regulation of hepatic bile acid synthesis, glycodeoxycholic acid (GDCA) was administered to 15 male and 14 female rabbits., Methods: After bile diversion, GDCA equivalent to the hepatic bile acid influx was infused intraduodenally. Biliary cholic acid output represented bile acid synthesis. Hepatic 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol 7 alpha-hydroxylase activities and steady state messenger RNA (mRNA) levels were determined., Results: GDCA inhibited bile acid synthesis less in female than in male rabbits. Hepatic HMG-CoA reductase activity decreased 39% in males, but increased 48% in females. Hepatic cholesterol 7 alpha-hydroxylase activity decreased similarly in males and females, and mRNA levels decreased 86% in males but were unchanged in females., Conclusions: (1) Total bile diversion stimulated both hepatic cholesterol and bile acid synthesis by activating the rate-controlling enzymes and increasing mRNA levels. (2) GDCA decreased mRNA levels of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in males, but mRNA levels did not decrease in females. (3) Bile acid synthesis was sustained in females because continued biosynthesis of cholesterol provided a substrate for cholesterol 7 alpha-hydroxylase and stimulus for enzyme formation.

The effect of ursodiol on the clinical and biochemical features, serum, urinary, and biliary bile acids was investigated over a 2-yr treatment period in 14 patients with primary biliary cirrhosis (stages II-IV). Pruritus and fatigue improved, and alkaline phosphatase and liver transferases declined significantly in all patients during therapy. In four patients, less inflammation was noted by liver biopsy after 2 yr, but histology of disease did not change. Serum and urinary bile acids were increased several-fold before treatment, with cholic acid predominating. Ursodiol accounted for 30% of biliary bile acids after administration (gallstone subjects approximately 50%), and was conjugated with glycine and taurine in a ratio of 7.3:1. However, in the endogenous bile acids, the ratio increased from 1.2:1 to only 2.1:1. About 6% unconjugated bile acids were secreted into the bile (healthy controls < 1%). Thus, in patients with primary biliary cirrhosis, a larger fraction of free bile acids and a higher proportion of taurine-conjugated bile acids are secreted into the bile, compared with healthy controls. Ursodiol improves symptoms and histology with lower biliary enrichment with this bile acid.

Feedback regulation of derepressed hepatic bile acid biosynthesis was studied individually with glycocholic, glycodeoxycholic, and glycoursocholic acids by infusion into bile acid-depleted rabbits. Construction of a bile fistula drained the endogenous bile acid pool (90% glycodeoxycholic acid, 10% glycocholic acid) within 24 hours and elicited maximal bile acid synthesis after about 72 hours, at which time glycocholic acid became the only biliary bile acid (greater than 98%). Replacement of the bile acid pool with glycocholic acid or glycodeoxycholic acid at a rate equivalent to the hepatic endogenous bile acid flux inhibited endogenous biosynthesis by 40%. In contrast, glycoursocholic acid, the 7 beta-hydroxy epimer of glycocholic acid, failed to suppress synthesis. Hepatic bile acid depletion increased hydroxymethyglutary coenzyme A (HMG-CoA) reductase activity fourfold and cholesterol 7 alpha-hydroxylase activity threefold, which were reduced 48% and 51%, respectively, from their maximum levels during replacement with glycocholic acid. Glycodeoxycholic acid infusion depressed cholesterol 7 alpha-hydroxylase activity by 59% without reducing HMG-CoA reductase activity significantly. There was no significant change in the activity of either enzyme during glycoursocholic acid infusion. Biliary cholesterol and cholestanol secretion declined 13% and 53%, respectively, during glycocholic acid infusion, were not affected by glycodeoxycholic acid infusion, but increased 19% and 43%, respectively, during glycoursocholic acid infusion. These results show that in rabbits the feedback regulation of hepatic bile acid synthesis depends on the hepatic flux of the normally present endogenous bile acids glycocholic acid and glycodeoxycholic acid but does not respond to the 7 beta-hydroxy glycoursocholic acid. Glycocholic acid inhibits both HMG-CoA reductase and cholesterol 7 alpha-hydroxylase while glycodeoxycholic acid affects primarily cholesterol 7 alpha-hydroxylase. Thus, the regulation of bile acid synthesis may be mediated by both the availability of cholesterol substrate and the activity of the rate-determining enzyme for bile acid synthesis.

Preparation of some biologically important keto bile acids is described. Advantage is taken of the preferential ketalization of 3-oxo group in bile acids over 7- and 12-oxo groups for the selective reduction of these keto groups. The method was found to be specially useful for preparation of 7 beta-, 12 alpha, and 12 beta-[3H]-3-oxo bile acids. Improved methods are also described for the preparation of epimers of naturally occurring bile acids at C-3, C-7, and C-12. 3 beta-Hydroxy bile acids (iso-bile acids) were prepared with the use of diethylazodicarboxylate/triphenylphosphine/formic acid. Iso-bile acids were obtained in excellent yields (80-95%) except during synthesis of isoursodeoxycholic acid (yield, 50%). Isoursodeoxycholic acid was, however, prepared in very good yield via epimerization of 3 alpha-hydroxyl group in 7-oxolithocholic acid followed by stereoselective reduction of 7-oxo group. A highly efficient method for the reduction of 7-oxo and 12-oxo groups was developed. Thus, 7-oxolithocholic acid and 7-oxoisolithocholic acid on reduction with potassium/tertiary amyl alcohol yielded ursodeoxycholic acid and isoursodeoxycholic acid in yields of 96% and 94%, respectively, while reduction of 7-oxodeoxycholic acid resulted in ursocholic acid in 93% yield. In a similar manner, reduction of 12-oxolithocholic acid and 12-oxochenodeoxycholic acid yielded 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid (lagodeoxycholic acid; 92% yield) and 3 alpha, 7 alpha, 12 beta-trihydroxy-5 beta-cholanoic acid (lagocholic acid, 86% yield).

The high-performance liquid chromatographic separation of the 25R and 25S diastereoisomers of the bile alcohols 5 beta-cholestane-3 alpha,7 alpha,26-triol and 5 beta-cholestane-3 alpha,7 alpha, 12 alpha, 26-tetrol and the bile acids, 3 alpha,7 alpha-dihydroxy-5 beta-cholestane-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane-26-oic acid is described. A Radial-Pak microBondapak C18 reversed-phase cartridge was used for the separations and elutions were carried out with acetonitrile-water-methanol-acetic acid mixtures. All eight diastereoisomeric compounds showed baseline separation when up to 200 micrograms of the isomeric mixtures were injected into the column and the method can be used for isolation of pure diastereoisomers of these bile acids and bile alcohols.

We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalytic efficiency. The administration of mevalonolactone, which is converted to cholesterol, modestly increased cholesterol 7 alpha-hydroxylase activity and enzyme mass in the bile acid-depleted rats. However, when taurocholate was infused together with mevalonolactone, cholesterol 7 alpha-hydroxylase activity and catalytic efficiency were markedly depressed while enzyme mass did not change as compared with bile acid-depleted rats. These results show that (a) hepatic bile acid depletion increases bile acid synthesis mainly by activating cholesterol 7 alpha-hydroxylase with only a small rise in enzyme mass, (b) replacement with taurocholate for 24 h decreases both cholesterol 7 alpha-hydroxylase activity and mass proportionally, (c) when cholesterol is available (mevalonolactone supplementation), the infusion of taurocholate results in the formation of a catalytically less active cholesterol 7 alpha-hydroxylase, and (d) tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, does not inhibit cholesterol 7 alpha-hydroxylase. Thus, bile acid synthesis is modulated by the catalytic efficiency and mass of cholesterol 7 alpha-hydroxylase. The enterohepatic flux of 7 alpha-hydroxylated bile acids and the formation of hepatic cholesterol apparently control cholesterol 7 alpha-hydroxylase by different mechanisms.

Cerebrotendinous xanthomatosis (CTX) is a rare recessive inherited lipid storage disease that was first described by Van Bogaert. Although the principal clinical presentation affects the nervous system with dementia, spinal cord paresis, cerebellar ataxia and peripheral neuropathy, the liver is is the organ where the major biochemical abnormalities are expressed. The following sections deal with the pathogenesis and treatment of the biochemical problems in CTX.

The effect of side chain conjugation on 7-dehydroxylation of bile acids has been investigated. C24-bile acids and their glycine and taurine conjugates and keto bile acids were incubated with pure strains of Eubacterium sp. VPI 12708. Bile acids of the 5 alpha- or 5 beta-series with a free terminal carboxyl group and a 3 alpha, 7 alpha-dihydroxy system were very effectively 7 alpha-dehydroxylated, whereas 7 beta-hydroxy bile acids resisted 7-dehydroxylation. Oxo bile acids were metabolized at the oxygen function also. Glycine- and taurine-conjugated bile acids were neither deamidated nor 7-dehydroxylated by the bacteria. Thus, side chain conjugation prevents 7-dehydroxylation of bile acids by Eubacterium sp. VPI 12708.

Guinea pig gallbladder bile contains chenodeoxycholic acid (62 +/- 5%), ursodeoxycholic acid (8 +/- 5%), and 7-ketolithocholic acid (30 +/- 5%). All three bile acids became labeled to the same specific activity within 30 min after [3H]cholesterol was injected into bile fistula guinea pigs. When a mixture of [3H]ursodeoxycholic acid and [14C]chenodeoxycholic acid was infused into another bile fistula guinea pig, little 3H could be detected in either chenodeoxycholic acid or 7-ketolithocholic acid. But, 14C was efficiently incorporated into ursodeoxycholic and 7-ketolithocholic acids. Monohydroxylated bile acids make up 51% and ursodeoxycholic acid 38% of fecal bile acids. After 3 weeks of antibiotic therapy, lithocholic acid was reduced to 6% of the total, but ursodeoxycholic acid (5-11%) and 7-ketolithocholic (15-21%) acid persisted in bile. Lathosterol constituted 19% of skin sterols and was detected in the feces of an antibiotic-fed animal. After one bile fistula guinea pig suffered a partial biliary obstruction, ursodeoxycholic and 7-ketolithocholic acids increased to 46% and 22% of total bile acids, respectively. These results demonstrate that chenodeoxycholic acid, ursodeoxycholic acid, and 7-ketolithocholic acid can all be made in the liver of the guinea pig.

An efficient and convenient procedure for the hydrolysis of bile acid methyl esters is described. This is achieved by the addition of aqueous lithium hydroxide in methanol/dioxane (or tetrahydrofuran) at room temperature. Under these conditions, the formates as well as the acetate derivatives were also hydrolyzed, and the desired bile acids were isolated in 86 to 94% yield.

We studied the effect of the orientation of the 7-hydroxyl group in taurocholate (7 alpha) and tauroursocholate (7 beta) on the feedback regulation of bile-acid synthesis and its rate-controlling enzyme, cholesterol 7 alpha-hydroxylase, in bile-fistula rats. To ensure a constant supply of cholesterol and to label newly synthesized bile acids, RS[2-14C]mevalonolactone was infused intraduodenally at 154 mumol/h before and during bile-acid infusion. Mevalonolactone inhibited hydroxymethyl-glutaryl CoA reductase activity 90% but did not increase bile-acid synthesis and cholesterol 7 alpha-hydroxylase activity. When sodium taurocholate was infused at the rate of 27 mumol/100 g rat per h (equivalent to the hourly hepatic bile-acid flux), bile-acid synthesis decreased 82% and cholesterol 7 alpha-hydroxylase activity declined 78%. This inhibitory effect was observed in the absence of hepatic damage. In contrast, sodium tauroursocholate infused at the same rate did not decrease bile-acid synthesis nor cholesterol 7 alpha-hydroxylase activity. Hepatic cholesterol content rose 36% with sodium taurocholate but did not change during sodium tauroursocholate administration. These results demonstrate that the feedback inhibition of bile-acid synthesis is mediated through the regulation of cholesterol 7 alpha-hydroxylase. In these experiments, taurocholate was a far more potent inhibitor than its 7 beta-hydroxy epimer, tauroursocholate.

We examined the relationship between cholesterol synthesis and high affinity low density lipoprotein (LDL) catabolism in freshly isolated mononuclear leukocytes and plasma sterols and apolipoprotein concentrations in three homozygous and one heterozygous subject with sitosterolemia with xanthomatosis and in 12 control subjects. Observations in untreated subjects were compared during therapy with lovastatin or interruption of the enterohepatic circulation of bile acids. Plasma cholesterol, plant sterol, and apolipoprotein B concentrations declined more than 50% in the two homozygous sitosterolemic subjects after ileal bypass surgery. In contrast, plasma cholesterol, plant sterol, and apolipoprotein B concentrations remained constant in a homozygous sitosterolemic subject and declined only 7% in a heterozygous sitosterolemic subject during 20 weeks of lovastatin (40 mg/day) treatment compared to a 28% decrease in similarly treated control subjects. Lovastatin treatment decreased cholesterol synthesis more than 60% but did not increase high affinity catabolism of LDL further in the sitosterolemic cells, compared to a more than 20% rise in control mononuclear leukocytes. Conversely, bile acid malabsorption increased cholesterol synthesis 59%, total hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity 13%, and receptor-mediated LDL degradation 41% in control cells, but did not stimulate cholesterol synthesis or microsomal HMG-CoA reductase activity in sitosterolemic mononuclear leukocytes although receptor-mediated LDL catabolism rose an additional 26%. These results demonstrate a greater than expected decrease in plasma sterols and apolipoprotein B concentrations in sitosterolemic subjects after stimulation of bile acid synthesis because of the inability to up-regulate cholesterol production. We suggest that bile acid-sequestering drugs or ileal exclusion surgery may be more effective treatments to mobilize accumulated sterol deposits and prevent atherosclerosis in this disease.

An improved method for the synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid is described. The method involves an Arndt-Eistert rearrangement of the corresponding diazoketone obtained by the action of diazoethane on 3 alpha, 7 alpha-diformyloxy-5 beta-cholane-24-carboxylic or 3 alpha, 7 alpha, 12 alpha-triformyloxy-5 beta-cholane-24-carboxylic acid chloride. The products are obtained in good yield and no isomeric 27-nor- 24-methyl acid contaminants are formed as encountered in the commonly employed Kolbe synthesis.

Orally administered UDCA dramatically reduces the secretion of cholesterol into the bile. During UDCA therapy cholesterol balance is maintained by a reduction in both the relative and absolute absorption of cholesterol and, perhaps, by a combined moderate enhancement of bile acid synthesis and a suppression of cholesterol production. The percentage of UDCA in the bile is limited by the inability of UDCA to suppress bile acid synthesis from cholesterol and by the conversion of UDCA to CDCA by the intestinal bacteria.

Because ursodeoxycholic and chenodeoxycholic acids are interconverted in humans via 7-ketolithocholic acid, bile acid metabolism was studied in the nutria (Myocastor coypus), the bile of which is known to contain these three bile acids. Relative concentrations of ursodeoxycholic (37% +/- 20%), 7-ketolithocholic (33% +/- 17%), and chenodeoxycholic (17% +/- 9%) acids in gallbladder bile were unchanged by 5-20 h of complete biliary diversion (n = 7). Injection of either [14C]cholesterol, [14C]ursodeoxycholic, [14C]7-ketolithocholic acid, or a mixture of [7 beta-3H]chenodeoxycholic acid and [14C]chenodeoxycholic acid into bile fistula nutria demonstrated that all three bile acids can be synthesized hepatically from cholesterol, that they are interconverted sparingly (2%-5%) by the liver, but that 7-ketolithocholic acid is an intermediate in the hepatic transformation of chenodeoxycholic acid to ursodeoxycholic acid. An animal that had been fed antibiotics showed an unusually elevated concentration of ursodeoxycholic acid in gallbladder and hepatic bile, suggesting that bacterial transformation of ursodeoxycholic acid in the intestine may be a source of some biliary chenodeoxycholic acid and 7-ketolithocholic acid.

The 7 alpha-hydroxylation of two cholesterol analogues, sitosterol and cholestanol, and their effect on the 7 alpha-hydroxylation of cholesterol were measured in rat and human hepatic microsomes. In untreated rat liver microsomes, the 7 alpha-hydroxylation of cholesterol was higher than that of cholestanol (1.4-fold) and sitosterol (30-fold). After removal of endogenous sterols from the microsomes by acetone treatment, the 7 alpha-hydroxylation of cholesterol was similar to that of cholestanol and only fourfold higher than that of sitosterol. Cholestanol and sitosterol competitively inhibited cholesterol 7 alpha-hydroxylase in both rat and human liver microsomes, with cholestanol the more potent inhibitor. Patients with sitosterolemia with xanthomatosis, who have elevated microsomal cholestanol and sitosterol, showed reduced cholesterol 7 alpha-hydroxylase activity relative to the activity in control subjects (13.9 and 14.7 vs. 20.3 +/- 0.9 pmol/nmol P-450 per min, P less than 0.01). Enzyme activity in these patients was 40% higher when measured in microsomes from which competing sterols had been removed. Ileal bypass surgery in one sitosterolemic patient decreased plasma cholestanol and sitosterol concentrations and resulted in a 30% increase in hepatic microsomal cholesterol 7 alpha-hydroxylase activity. Cholesterol 7 alpha-hydroxylase appears to have a specific apolar binding site for the side chain of cholesterol and is affected by the presence of cholestanol and sitosterol in the microsomal substrate pool. Reduced bile acid synthesis in sitosterolemia with xanthomatosis may be related to the inhibition of cholesterol 7 alpha-hydroxylase activity by endogenous cholesterol analogues.

We have examined the mechanism for the bacterial transformation of chenodeoxycholic acid and lithocholic acid into the corresponding 3 beta-hydroxy epimers with the use of 3 alpha- and 3 beta-tritiated bile acids. The 3-oxo bile acids were transformed into the 3 alpha- (85%) and 3 beta- (15%) hydroxy bile acids after 20-hr incubation with Clostridium perfringens. Approximately 75% radioactivity was recovered in the aqueous medium when [3 beta-3H]chenodeoxycholic acid or [3 beta-3H]lithocholic acid was incubated with the bacteria, and approximately 15% of radioactivity in the bile acid fraction was associated with the 3 alpha-position of the iso-bile acids. When [3 beta-3H]chenodeoxycholic acid was incubated with unlabeled 3-oxo-5 beta-cholanoic acid, tritiated litho- and iso-lithocholic acids were recovered. These results can be explained only when a 3-oxo intermediate is postulated, and the 3 beta-hydrogen in the bile acids is transferred by the bacterial coenzyme (NAD+ or NADP+) to the 3 alpha-position in the iso-bile acids during the reduction of the 3-oxo compounds.

The effect of 7-ketolithocholic acid on biliary bile acid composition, cholesterol saturation, and as an intermediate in the conversion of chenodeoxycholic acid to ursodeoxycholic acid was investigated in 5 subjects with gallstones. After 7-ketolithocholic acid (400 mg/day) was administered orally for 14 days, biliary bile acid composition changed: The proportion of cholic acid decreased (from 45% to 19%), deoxycholic acid decreased (from 15% to 10%), chenodeoxycholic acid increased markedly (from 36% to 59%), ursodeoxycholic acid increased (from 36% to 59%), ursodeoxycholic acid increased (from 2% to 7%), and lithocholic acid increased (from 2% to 5%), while only trace amounts of 7-ketolithocholic acid were detected. During this treatment, the biliary lithogenic index fell from 2.6 to 0.9 and was accompanied by a pronounced drop in biliary cholesterol concentration. After biliary bile acid levels became constant [24-14C]chenodeoxycholic acid was given intravenously as a pulse-label, and the resultant biliary ursodeoxycholic acid and lithocholic acid specific activity curves showed a precursor--product relationship with chenodeoxycholic acid. Similarly, when uniformly labeled 7-[24-14C]ketolithocholic acid was fed (400 mg/day, 1000 +/- 100 dpm/mg) the specific activities of biliary chenodeoxycholic acid and ursodeoxycholic acid became constant and approximated each other, but these were only 75% as high as the fed 7-ketolithocholic acid. These results indicate that 7-ketolithocholic acid is absorbed, and suppresses endogenous bile acid production and biliary cholesterol secretion. Both isotopic experiments infer that ursodeoxycholic acid and lithocholic acid are formed from chenodeoxycholic acid and not from 7-ketolithocholic acid. The reduction in biliary lithogenic index and in cholesterol concentration suggest that low doses of 7-ketolithocholic acid may be effective in dissolving gallstones.

In order to determine whether cholestanol and bile acids are derived from the same precursor, key intermediates of both biosynthetic pathways beyond cholesterol were administered intravenously to a patient with cerebrotendinous xanthomatosis and to a control subject. After pulse-labeling with [4-14C]4-cholesten-3-one and [G-3H]7 alpha-hydroxy-4-cholesten-3-one, cholestanol, cholesterol, and the two primary bile acids, cholic acid and chenodeoxycholic acid were isolated from specimens of bile. Each compound was purified by thin-layer chromatography and conclusively identified by gas-liquid chromatography-mass spectrometry. In other studies, the in vitro formation of 4-cholesten-3-one from cholesterol was measured in hepatic microsomal fractions prepared from a subject with cerebrotendinous xanthomatosis and from 3 control individuals. In all subjects, cholic acid and chenodeoxycholic acid were labeled with tritium, but neither cholesterol nor cholestanol contained this isotope. In contrast, 14C was detected in the cholestanol fraction with trace amounts in chenodeoxycholic acid, cholic acid, and cholesterol. Hepatic microsomes prepared from liver biopsy specimens obtained from a subject with cerebrotendinous xanthomatosis produced three times more 4-cholesten-3-one than the controls. The results indicate that 4-cholesten-3-one was converted primarily into cholestanol and 7 alpha-hydroxy-4-cholesten-3-one into cholic acid and chenodeoxycholic acid. Neither ketonic steroid was transformed into cholesterol. The increased production of cholestanol in cerebrotendinous xanthomatosis may be accounted for by enhanced hepatic formation of 4-cholesten-3-one. 7 alpha-Hydroxy-4-cholesten-3-one is a precursor of bile acids, but not of cholestanol.

To examine the defect in side-chain oxidation during the formation of bile acids in cerebrotendinous xanthomatosis, we measured in vitro hepatic microsomal hydroxylations at C-12 and C-25 and mitochondrial hydroxylation at C-26 and related them to the pool size and synthesis rates of cholic acid and chenodeoxycholic acid as determined by the isotope dilution technique. Hepatic microsomes and mitochondria were prepared from seven subjects with cerebrotendinous xanthomatosis and five controls. Primary bile acid synthesis was markedly reduced in cerebrotendinous xanthomatosis as follows: cholic acid, 133 +/- 30 vs. 260 +/- 60 mg/d in controls; and chenodeoxycholic acid, 22 +/- 10 vs. 150 +/- 30 mg/d in controls. As postulated for chenodeoxycholic acid synthesis, mitochondrial 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol was present in all specimens and was 30-fold more active than the corresponding microsomal 25-hydroxylation. However, mean mitochondrial 26-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol was less active in cerebrotendinous xanthomatosis than in controls: 59 +/- 17 compared with 126 +/- 21 pmol/mg protein per min. As for cholic acid synthesis, microsomal 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was substantially higher in cerebrotendinous xanthomatosis and control preparations (620 +/- 103 and 515 +/- 64 pmol/mg protein per min, respectively) than the corresponding control mitochondrial 26-hydroxylation of the same substrate (165 +/- 25 pmol/mg protein per min). Moreover in cerebrotendinous xanthomatosis, mitochondrial 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol-26-hydroxylase activity was one-seventh as great as in controls. Hepatic microsomal 12 alpha-hydroxylation, which may be rate-controlling for the cholic acid pathway, was three times more active in cerebrotendinous xanthomatosis than in controls: 1,600 vs. 500 pmol/mg protein per min. These results demonstrate severely depressed primary bile acid synthesis in cerebrotendinous xanthomatosis with a reduction in chenodeoxycholic acid formation and pool size disproportionately greater than that for cholic acid. The deficiency of chenodeoxycholic acid can be accounted for by hyperactive microsomal 12 alpha-hydroxylation that diverts precursors into the cholic acid pathway combined with decreased side-chain oxidation (mitochondrial 26-hydroxylation). However, side-chain oxidation in cholic acid biosynthesis may be initiated via microsomal 25-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol was substantially lower in control and cerebrotendinous xanthomatosis liver. Thus, separate mechanisms may exist for the cleavage of the cholesterol side chain in cholic acid and chenodeoxycholic acid biosynthesis.

Large amounts of cholestanol, the 5 alpha-dihydro derivative of cholesterol are found in tissues of patients with the rare inherited sterol storage disease cerebrotendinous xanthomatosis. Although small amounts of cholestanol are present in virtually every tissue of normal man, little is known about its metabolism and effect on cholesterol and bile acid formation. The purpose of this study is to investigate the absorption and metabolism of cholestanol and its early effects on hepatic morphology and on the rate-limiting enzymes of cholesterol and bile acid biosynthesis. After 2 wk on a diet supplemented with 2% cholestanol, total liver sterol content increased by 48% (3.26 vs. 2.20 mg/g), and resulted in a significant rise in hepatic cholestanol concentration to 1.4 mg/g. However, cholestanol was less efficiently absorbed from the intestine than cholesterol and interfered with cholesterol absorption. Furthermore, hepatic hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase activity rose 2.6-fold (from 150.3 to 397.0 pmol/mg per min) during cholestanol feeding, and was associated with a marked proliferation of the smooth endoplasmic reticulum of the centrilobular areas. In addition, significant amounts of allocholic acid (16%) and allochenodeoxycholic acid (5%) were formed from cholestanol and excreted in the bile. These results show that cholestanol is absorbed from the intestine, interferes with cholesterol absorption, and is deposited in the liver. However, in contrast to cholesterol, cholestanol feeding was associated with a marked elevation of HMG-CoA reductase activity. Thus, despite structural similarity between cholesterol and its 5 alpha-saturated derivative, cholestanol does not exert feedback inhibition on hepatic cholesterol biosynthesis.

We have compared the effect of ursodeoxycholic acid with placebo on the clinical state, blood liver chemistries and serum and urinary bile acids in four patients with primary biliary cirrhosis. All parameters were evaluated monthly, and bile acid composition was measured by capillary gas-liquid chromatography. At the time of admission, all patients showed intense pruritus, and their serum alkaline phosphatase, AST and ALT levels were elevated 4.3, 2.7 and 2.3 times over control values. Serum bile acids were elevated almost 38-fold with 2.5 times more cholic acid than chenodeoxycholic acid. Urinary bile acid output was elevated 28 times the control values, and 36% were 1 beta-hydroxycholic acid, 1 beta-hydroxydeoxycholic acid and hyocholic acid (3 alpha,6 alpha, 7 alpha-trihydroxy-5 beta-cholanoic acid). Three months of placebo administration did not significantly affect the clinical or biochemical presentations, and the serum and urinary bile acid composition did not change. In contrast, ursodeoxycholic acid feeding (12 to 15 mg per kg per day) for 6 months abolished pruritus in two and lessened itching in two subjects and reduced serum alkaline phosphatase, AST and ALT levels by 21, 35 and 47%, respectively. The mean values for the total serum bile acid concentrations in these patients declined 26% from the pretreatment value, but the proportion of ursodeoxycholic acid increased from 3 to 40% of the total bile acids; thus, total fasting serum endogenous bile acid levels decreased almost 50%. Similar changes were noted in the urinary bile acids, in which ursodeoxycholic acid became the major bile acid, and approximately 18% were hydroxylated at C-1, C-6 and C-21.(ABSTRACT TRUNCATED AT 250 WORDS)