Search

Recent studies have suggested that bile acid acyl glucuronides form covalently bound protein adducts which may cause hypersensitivity reactions and increased morbidity in patients. Although the preferential biosynthesis of the acyl glucuronides has been known, the characterization of hepatic bile acid acyl glucuronosyltransferase has not yet been clearly elucidated. We have investigated the substrate specificity of the hepatic bile acid acyl glucuronosyltransferase using five common bile acids as substrates. The glucuronidation rate was dependent on the number of the hydroxy group on the steroid nucleus and mono-hydroxylated lithocholic acid, the more lipophilic common bile acid, was most effectively metabolized into its acyl glucuronide. The tri-hydroxylated cholic acid, the more water-soluble common bile acid, barely transformed into its glucuronide. Results showed decreasing of the initial velocity of the acyl glucuronidation with increasing of the concentration of substrate, lithocholic acid, owing to the substrate inhibition of the hepatic bile acid acyl glucuronosyltransferase. The substrate analogues, glycine and taurine conjugated bile acids, which exist in the body fluids in high concentrations, also inhibited the enzyme's activity. In addition, enzymatic reaction products, bile acid acyl glucuronides, also inhibited the activity. These inhibitory mechanisms may be responsible for the low concentration of bile acid acyl glucuronides in urine and may be an important detoxification system in the body.

Background: Deoxycholic acid (DCA), implicated in the pathogenesis of gall stones and colorectal cancer, is mainly formed by bacterial deconjugation (cholylglycine hydrolase (CGH)) and 7 alpha-dehydroxylation (7 alpha-dehydroxylase (7 alpha-DH)) of conjugated cholic acid (CA) in the caecum/proximal colon. Despite this, most previous studies of CGH and 7 alpha-DH have been in faeces rather than in caecal contents. In bacteria, CA increases 7 alpha-DH activity by substrate-enzyme induction but little is known about CA concentrations or CA/7 alpha-DH induction in the human colon., Aims and Methods: Therefore, in fresh "faeces", and in caecal aspirates obtained during colonoscopy from 20 patients, we: (i) compared the activities of CGH and 7 alpha-DH, (ii) measured 7 alpha-DH in patients with "low" and "high" percentages of DCA in fasting serum (less than and greater than the median), (iii) studied CA concentrations in the right and left halves of the colon, and examined the relationships between (iv) 7 alpha-DH activity and CA concentration in caecal samples (evidence of substrate-enzyme induction), and (v) 7 alpha-DH and per cent DCA in serum., Results: Although mean CGH activity in the proximal colon (18.3 (SEM 4.40) x10(-2) U/mg protein) was comparable with that in "faeces" (16.0 (4.10) x10(- 2) U/mg protein), mean 7 alpha-DH in the caecum (8.54 (1.08) x10(-4) U/mg protein) was higher (p<0.05) than that in the left colon (5.72 (0.85) x10(-4) U/mg protein). At both sites, 7 alpha-DH was significantly greater in the "high" than in the "low" serum DCA subgroups. CA concentrations in the right colon (0.94 (0.08) micromol/ml) were higher than those in the left (0.09 (0.03) micromol/ml; p<0.001) while in the caecum (but not in the faeces) there was a weak (r=0.58) but significant (p<0.005) linear relationship between 7 alpha-DH and CA concentration. At both sites, 7 alpha-DH was linearly related (p<0.005) to per cent DCA in serum. INTERPRETATION/SUMMARY: These results: (i) confirm that there are marked regional differences in bile acid metabolism between the right and left halves of the colon, (ii) suggest that caecal and faecal 7 alpha-DH influence per cent DCA in serum (and, by inference, in bile), and (iii) show that the substrate CA induces the enzyme 7 alpha-DH in the caecum.

Butyrate is derived from the microbial metabolism of dietary fiber in the colon where it plays an important role in linking colonocyte turnover and differentiation to luminal content. In addition, butyrate appears to have both anti-inflammatory and cancer chemopreventive activities. Using confocal microscopy and cell fractionation studies, butyrate pretreatment of a human colon cell line (HT-29 cells) inhibited the tumor necrosis factor-alpha (TNF-alpha)-induced nuclear translocation of the proinflammatory transcription factor NF-kappaB. Butyrate inhibited NF-kappaB DNA binding within 30 min of TNF-alpha stimulation, consistent with an inhibition of nuclear translocation. IkappaB.NF-kappaB complexes extracted from butyrate-treated cells were relatively resistant to in vitro dissociation by deoxycholate, suggesting a change in cellular IkappaB composition. Butyrate treatment increased p100 expression, an IkappaB that was not degraded upon TNF-alpha treatment. Butyrate also reduced the extent of TNF-alpha-induced IkappaB-alpha degradation and enhanced the presence of ubiquitin-conjugated IkappaB-alpha. The suppression of IkappaB-alpha degradation corresponded with a reduction in cellular proteasome activity as determined by in vitro proteasome assays and the increased presence of ubiquitin-conjugated proteins. The butyrate suppression of IkappaB-alpha degradation and proteasome activity may derive from its ability to inhibit histone deacetylases since the specific deacetylase inhibitor trichostatin A had similar effects. These results suggest a potential mechanism for the anti-inflammatory activity of butyrate and demonstrate the interplay between short chain fatty acids and cellular proteasome activity.

Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of BSSL on bile acids for activity with water-insoluble substrates differentiates it from other lipases. We have determined the crystal structure of a truncated variant of human BSSL (residues 1-5.8) and refined it at 2.60 A resolution, to an R-factor of 0.238 and R(free) of 0.275. This variant lacks the C-terminal alpha-helix and tandem C-terminal repeat region of native BSSL, but retains full catalytic activity. A short loop (residues 115-126) capable of occluding the active-site (the active site loop) is highly mobile and exists in two conformations, the most predominant of which leaves the active-site open for interactions with substrate. The bile salt analogue 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid (CHAPS) was present in the crystallisation medium, but was not observed bound to the enzyme. However, the structure reveals a sulfonate group from the buffer piperizine ethane sulfonic acid (PIPES), making interactions with Arg63 and His115. His115 is part of the active-site loop, indicating that the loop could participate in the binding of a sulphate group from either the glycosaminoglycan heparin (known to bind BSSL) or a bile acid such as deoxycholate. Opening of the 115-126 active-site loop may be cooperatively linked to a sulphate anion binding at this site. The helix bundle domain of BSSL (residues 319-398) exhibits weak electron density and high temperature factors, indicating considerable structural mobility. This domain contains an unusual Asp:Glu pair buried in a hydrophobic pocket between helices alpha(H) and alpha(K) that may be functionally important. We have also solved the structure of full-length glycosylated human BSSL at 4.1 A resolution, using the refined coordinates of the truncated molecule as a search model. This structure reveals the position of the C-terminal helix, missing in the truncated variant, and also shows the active-site loop to be in a closed conformation., (Copyright 2001 Academic Press.)

Background and Aims: Octreotide treatment of acromegalic patients induces cholesterol gallstone formation, in part by impairing cholecystokinin release and gall-bladder contraction. However, there are few data on the effect of octreotide on biliary arachidonic acid-rich phospholipids or mucin glycoprotein, factors which also influence cholesterol gallstone formation., Methods: In acromegalic patients studied before and during 3 months of octreotide treatment, we measured mucin glycoprotein concentrations and the molecular species of phosphatidylcholine, and related the results to the cholesterol saturation and percentage of deoxycholic acid in gall-bladder bile., Results: The relative proportions of the major arachidonic acid-rich phosphatidylcholine species, PC 16:0-20:4 and PC 18:0-20:4, increased significantly during octreotide treatment. These changes were associated with a rise in the cholesterol saturation index and a non-significant twofold increase in mucin glycoprotein concentration. There were significant correlations between PC 16:0-20:4 and the cholesterol saturation index, percentage of vesicular cholesterol and percentage of deoxycholic acid in gall-bladder bile., Conclusions: In acromegalic patients, octreotide increases the proportions of arachidonic acid-rich phospholipids, with associated rises in: (a) the cholesterol saturation index and percentage of vesicular cholesterol, and (b) the percentage of deoxycholic acid in gall-bladder bile-changes similar to those found in patients with cholesterol-rich gall-bladder stones.

Prolonged large bowel transit time and an associated increase in the proportion of deoxycholic acid (DCA) in serum and bile have been implicated in the development of cholesterol-rich gallstones and colon cancer. Prolongation of intestinal transit also increases intracolonic pH that, we hypothesized, should favor the solubilization and absorption of newly formed DCA within the colon. To test this hypothesis, we performed in vitro studies on homogenized cecal aspirates (obtained at colonoscopy) that were incubated anaerobically with [14C]cholic acid for 16 h after which the pH was adjusted to between 4.0 and 7.0 in 0.5-pH unit steps. The resultant reaction mixtures were centrifuged to separate the supernatant from the precipitate, and the specific activity of [14C]DCA was quantitated in both phases. As the pH in the aspirates was manipulated from 4.0 to 7.0, the proportion of newly formed, labeled DCA increased in the supernatant and fell in the precipitate, particularly at a hydrogen ion concentration of <100 x 10(-7) (equivalent to pH 5.0-7.0). These results show that the solubility of DCA in colonic contents increases with increasing pH. If solubility is rate limiting, this should lead to increased absorption that, in turn, would explain why the proportion of DCA in serum and bile increases with the prolongation of large bowel transit time.

We studied the effects of deoxycholic acid and its three epimers with beta-hydroxyl groups (3alpha,12beta-, 3beta,12alpha-, and 3beta,12beta-dihydroxy-5beta-cholan-24-oic acids), which were hydrophilic and less cytotoxic, on lipid peroxidation to elucidate the relationship between structural features of bile acids and their effect on lipid peroxidation. Taurodeoxycholate markedly increased the production of thiobarbituric acid-reactive substances, end products of lipid peroxidation, in isolated rat hepatocytes, whereas epimers of taurodeoxycholate did not. Deoxycholic acid inhibited mitochondrial NADH dehydrogenase and NADH:ferricytochrome c oxidoreductase activities, leading to free radical generation, whereas epimers of deoxycholic acid had no effect on mitochondrial enzymes. These findings suggested that hydrophobic bile acids cause lipid peroxidation by impairment of mitochondrial function, leading to the generation of free radicals; and epimerization of alpha-hydroxyl groups in the steroid nucleus to beta-hydroxyl groups results in a decrease of the toxic effects of deoxycholic acid on lipid peroxidation.

The goal of this investigation was to establish a fast method to screen various insulin absorption enhancers by following their effect on the initial kinetics of insulin incubated with alpha-chymotrypsin at 37 degrees C. A simple, sensitive and reproducible reversed phase high performance liquid chromatography (HPLC) method has been developed to carry out this goal. Linear responses (r > 0.999) were observed over the range of 0.4-4 U/ml for insulin. There was no significant difference (P < 0.05) between inter- and intra-day studies for insulin. The mean relative standard deviations (RSD%) of the results of within-day precision and accuracy of insulin were 12%. The assay was sensitive to detect the existence of any metabolite due to the addition of any absorption enhancers, even if it was not seen with insulin alone. Three metabolites (A-C) were detected only when insulin was incubated with alpha-chymotrypsin at 37 degrees C. Metabolite D was observed when either glycocholic acid (0.5, 1%) or taurochenodeoxycholate (0.5, 1%) was incubated with insulin in the absence of alpha-chymotrypsin at 37 degrees C. The compounds that significantly increased insulin T50% were glycyrrhizic acid (0.5%) > deoxycholic acid (1%) > deoxycholic acid (0.5%) > glycyrrhizic acid (1%) > cholic acid (0.5, 1%). Capric acid (0.5%), hydroxypropyl-alpha-cyclodextrin (0.5, 1%) and dimethyl-alpha-cyclodextrin (0.5, 1, 5%) did not significantly affect insulin T50%. The bile salts increased insulin T50% in this order: deoxycholate > cholate > glycocholate > taurocholate > taurodeoxycholate > taurochenodeoxycholate > glycodeoxycholate. The results obtained would support the feasibility of utilizing such method for screening any compound incorporated in insulin formulation. These compounds should be used in the minimum possible concentration to avoid or minimize insulin degradation.

The quantitation of cholesterol in lipoprotein subfractions is valuable in estimating the risk for coronary artery disease, but requires multiple tests. We describe a relatively simple procedure, referred to as the dual HDL/total cholesterol (DHT) assay, which allows the sequential measurement of HDL cholesterol (HDL-C) and total cholesterol (total-C) in a single tube. HDL-C is first measured using a homogeneous assay that utilizes an anti-apolipoprotein B [apo(B)] antibody, which sterically blocks the enzymatic measurement of cholesterol on the non-HDL subfractions. Next, deoxycholate is added, which disrupts the antibody-apo(B) complex and allows the subsequent enzymatic measurement of the remaining cholesterol in the non-HDL subfractions. The DHT assay has an acceptable analytical performance and yields results similar to standard methods: for HDL-C, y(DHT) = 0.98x + 0.19, r=0.90; for total-C, y(DHT) = 1.11x - 0.09, r=0.99. In summary, the DHT assay is a homogeneous assay for both HDL-C and total-C, and provides a simple and cost-effective method for screening for hyperlipidaemia.

Deoxycholic acid (DCA) has long been implicated as tumour-promoting agent in the colon. Polyamines are necessary for cell proliferation, they are accumulated in high amounts in colon cancer cells, and their concentrations in the colonic lumen can reach millimolar levels. The aim of this study was to investigate the effects of physiological DCA concentrations on proliferation and polyamine content in human colon cancer cells (Caco-2) in culture. Over an initial 48 h in culture, DCA stimulated Caco-2 cell proliferation rate three-fold, reaching a maximum with 20 microM DCA. DCA-induced increases in ornithine decarboxylase (ODC) activity corresponded to peak proliferation rates, occurring only during the initial 48 h of cell proliferation. Treatment with low-dose DCA resulted in a two-fold increase in putrescine uptake, first noted after 2 days in culture, but persisting until the cells became confluent (day 5). Both basal and DCA-stimulated putrescine uptake in Caco-2 cells were saturable. Kinetic analysis of the uptake data showed that DCA-stimulated putrescine uptake was due to an increase in the capacity of the putative putrescine transporter, without changes in its affinity, therefore implying an increased number of putrescine transporters in the cell membrane, without change in their structure.

The aim of this article is to review selected aspects of the pathogenesis of cholesterol-rich, gall-bladder stones (GBS)--with emphasis on recent developments in biliary cholesterol saturation, cholesterol microcrystal nucleation, statis within the gall-bladder and, particularly, on the roles of intestinal transit and altered deoxycholic acid (DCA) metabolism, in GBS development. In biliary cholesterol secretion, transport and saturation, recent developments include evidence in humans and animals, that bile lipid secretion is under genetic control. Thus in mice the md-2 gene, and in humans the MDR-3 gene, encodes for a canalicular protein that acts as a 'flippase' transporting phospholipids from the inner to the outer hemi-leaflet of the canalicular membrane. In the absence of this gene, there is virtually no phospholipid or cholesterol secretion into bile. Furthermore, when inbred strains of mice that have 'lith genes' are fed a lithogenic diet, they become susceptible to high rates of GBS formation. The precipitation/nucleation of cholesterol microcrystals from supersaturated bile remains a critical step in gallstone formation. methods of studying this phenomenon have now been refined from the original 'nucleation time' to measurement of cholesterol appearance/detection times, and crystal growth assays. Furthermore, the results of recent studies indicate that, in addition to classical Rhomboid-shape monohydrate crystals, cholesterol can also crystallize, transiently, as needle-, spiral- and tubule-shaped crystals of anhydrous cholesterol. A lengthy list of promoters, and a shorter list of inhibitors, has now been defined. There are many situations where GB stasis in humans is associated with an increased risk of gallstone formation--including iatrogenic stone formation in acromegalic patients treated chronically with octreotide (OT). As well as GB stasis, however, OT-treated patients all have 'bad' bile which is supersaturated with cholesterol, has excess cholesterol in vesicles, rapid microcrystal mulceation times and a two-fold increase in the percentage DCA in bile. This increase in the proportion of DCA seems to be due to OT-induced prolongation of large bowel transit time (LBTT). Thus LBTT is linearly related to (i) the percentage of DCA in serum; (ii) the DCA pool size; and (III) the DCA input or 'synthesis' rate. Furthermore, the intestinal prokinetic, cisapride, counters the adverse effects of OT on intestinal transit, and 'normalizes' the percentage of DCA in serum/bile. Patients with spontaneous gallstone disease also have prolonged LBTTs, more colonic gram-positive anaerobes, increased bile acid metabolizing enzymes and higher intracolonic pH values, than stone-free controls. Together, these changes lead to increased DCA formation, solubilization and absorption, Thus, in addition to the 'lithogenic liver' and 'guilty gall-bladder' one must now add the 'indolent intestine' to the list of culprits in cholesterol gallstone formation.

A method for the determination of the activity of hepatic glucuronyltransferase catalyzing formation of bile acid 24-glucuronides using high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD) has been developed. Bile acid 24-glucuronides were simultaneously separated on a semimicrobore column, Capcell Pak C18UG120, using 20 mM ammonium phosphate (pH 6.0)-acetonitrile (27:10 and 16:10) as the mobile phase in the stepwise gradient elution mode. A 1 M potassium hydroxide solution for the hydrolysis of the 24-glucuronides, which liberates the corresponding bile acids and glucuronic acid, was mixed with the mobile phase in a post-column mode, and the resulting eluant was heated at 90 degrees C, the 24-glucuronides being monitored using a pulsed amperometric detector; the limit of detection was 10 ng. The proposed method was applied to the determination of the hepatic enzyme activity catalyzing bile acid 24-glucuronide formation and the result exhibited the efficient 24-glucuronide formation of the monohydroxylated bile acid, lithocholic acid.

Background/aims: The development of cholesterol gallstones, in some patients, has been associated with increased proportions of deoxycholic acid in the bile acid pool. Deoxycholic acid is a microbial product of cholic acid 7alpha-dehydroxylation in the intestines. The levels and activities of bile acid 7alpha-dehydroxylating bacteria have been reported to be increased in gallstone patients. The aim of the current study was to isolate 7alpha-dehydroxylating bacteria from gallstone patients and determine if these individuals are colonized by similar bacterial species., Methods: The levels of 7alpha-dehydroxylating bacteria in fecal samples were determined by fecal dilutions in 24 gallstone patients and 10 controls. 7alpha-Dehydroxylating bacteria were isolated by a non-selective streak plate technique and 7alpha-dehydroxylation activity was determined by measuring the conversion of [14C]-cholic acid to [14C]-deoxycholic acid using thin-layer chromatography., Results: Gallstone patients had >42-fold (p<0.01) higher levels of 7alpha-dehydroxylating bacteria than patients who had not developed gallstones. Eighteen strains of 7alpha-dehydroxylating bacteria were isolated from eight gallstone patients. Attempts to isolate 7alpha-dehydroxylating bacteria from ten control patients were unsuccessful using identical isolation techniques. Surprisingly, all strains of bacteria isolated from gallstone patients appear to belong to the genus Clostridium., Conclusion: Gallstone patients have higher levels of 7alpha-dehydroxylating fecal bacteria and appear to harbor only members of the genus Clostridium with this activity.

High levels of bile acids in the colon may correlate with an increased risk of colon cancer, but the underlying mechanisms are not known. Proteoglycan structures have been shown to change when human colon cells differentiate in vitro. The expression of [(35)S]sulphated molecules was used as a phenotypic marker to study the effects of bile acids on the human-colon-carcinoma cell line CaCo-2. [(35)S]sulphated compounds were isolated from the medium of cell fractions of cells metabolically labelled with [(35)S]sulphate in the absence and presence of cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid (LA). Labelled molecules were analysed by gel chromatography, HPLC and SDS/PAGE in combination with chemical and enzymic methods. The expression of (35)S-labelled proteoglycans was not affected by any of the bile acids tested. However, the level of sulphated metabolites increased 7-18-fold in different experiments during a 22 h labelling period in the presence of an LA concentration of 10 microg/ml (26.6 nmol/ml) compared with controls. Further analyses showed that this was due, at least in part, to the sulphation of LA itself. This sulphation of LA was a rapid process followed by secretion back to the medium. Brefeldin A did not reduce the sulphation of LA, indicating that this conversion takes place in the cytosol, rather than in the Golgi apparatus of the CaCo-2 cells. LA in colon may be sulphated efficiently by the colonocytes to reduce the toxic effects of this particular bile acid. Sulphation may possibly be an important protective mechanism in the colon.

Linoleic and arachidonic acids were inserted into phosphatidylcholine deoxycholate mixed micelles (PDM-micelles) with their tail groups buried inside and carboxylic groups exposed outside. The fatty acid hydrophobic tail had a high affinity for the hydrophobic region of phosphatidylcholine micelles. The fatty acids inserted into phosphatidylcholine micelles were better substrates for soybean lipoxygenase 1 (LOX1) with two distinct pH optima at 7.0 and 10.0. With Tween 20-solubilized linoleic acid, the enzyme had a pH optimum at 9.0, exclusively forming 13-hydroperoxides. However, with linoleic and arachidonic acids inserted into PDM-micelles, LOX1 synthesized exclusively 9- and 5-hydroperoxides, respectively. The enzyme brought about the transformation of the substrate either at pH 7.4 or at 10.0, less efficiently at pH 10.0. However, the regioselectivity of the enzyme was not altered by increasing the pH from 7.4 to 10.0. Thus, LOX1 could utilize fatty acids bound to membranes as physiological substrates. The enzyme utilized the carboxylic group of linoleic and arachidonic acids inserted into the PDM-micelles as a recognition site to convert the compounds into 9- and 5-hydroperoxides, respectively. This was confirmed by activity measurements using methyl linoleate as the substrate. Circular dichroism measurement of LOX1 with PDM-micelles suggested that while there was a small change in the tertiary structure of LOX1, the secondary structure was unaffected. Soybean LOX1, which is arachidonate 15-LOX, acted as "5-LOX", thus making it possible to change the regiospecificity of the LOX1-catalyzed reaction by altering the physical state of the substrate.

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3alpha,6beta, 7beta-trihydroxy bile acid beta-muricholic acid into hyodeoxycholic acid (3alpha,6alpha-dihydroxy acid) by dehydroxylation of the 7beta-hydroxy group and epimerization of the 6beta-hydroxy group into a 6alpha-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3alpha, 6alpha,7alpha-trihydroxy acid), alpha-muricholic acid (3alpha,6beta, 7alpha-trihydroxy acid), and omega-muricholic acid (3alpha,6alpha, 7beta-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted beta-muricholic acid almost completely into hyodeoxycholic acid.

Hepatic bile acid synthesis is regulated by recirculating bile acids, possibly by modulating the availability of newly synthesized and preformed cholesterol. Because data in the hamster on this mechanism are lacking, we fitted these animals with an extracorporeal bile duct and administered tritiated water intraperitoneally to label newly formed cholesterol. After interruption of the enterohepatic circulation, physiological and double-physiological doses of conjugated cholate (25 or 50 micromol/100 g. h) or of unconjugated deoxycholate (6 or 12 micromol) were infused intraduodenally for 54 hours and compared with controls. De novo and preformed cholesterol directly secreted into bile or used for cholate and chenodeoxycholate synthesis were quantitated by high-pressure liquid chromatography (HPLC)-liquid scintillation. Directly after depletion of the bile acid pool (6-9 hours) at nearly physiological conditions, chenodeoxycholate synthesis was significantly reduced by cholate and deoxycholate by up to 45% to 51%, whereas cholate formation decreased by approximately 22% during deoxycholate. This short-term effect was mainly mediated by reduced synthesis from preformed cholesterol. After long-term bile depletion (30-54 hours), bile acid synthesis returned to control levels during 25 micromol of cholate and of both deoxycholate doses. In contrast, only 50 micromol of cholate prevented derepression of bile acid synthesis. This long-term effect was mainly attributed to a diminished formation from de novo cholesterol exceeding the reduced synthesis from preformed cholesterol. In summary, short- and long-term regulation of bile acid synthesis in hamsters differs with respect to availabilities of preformed and de novo cholesterol.

Bile acids regulate the transcription of genes that control cholesterol homeostasis through molecular mechanisms that are poorly understood. Physiological concentrations of free and conjugated chenodeoxycholic acid, lithocholic acid, and deoxycholic acid activated the farnesoid X receptor (FXR; NR1H4), an orphan nuclear receptor. As ligands, these bile acids and their conjugates modulated interaction of FXR with a peptide derived from steroid receptor coactivator 1. These results provide evidence for a nuclear bile acid signaling pathway that may regulate cholesterol homeostasis.

The adjuvanticity of lamellar particles of poly(L-lactide) (PLA) towards adsorbed ovalbumin (OVA) was investigated. The aim of vaccine formulation was to maximise the amount of antigen retained on the particles and the time of retention during incubation of the formulations in PBS at 37 degrees C. Unmodified PLA lamellae were capable of adsorbing large quantities of OVA (up to 12.5% w/w) but major and rapid desorption occurred in PBS at 37 degrees C (80% released in 24 h). Retention of OVA on PLA lamellae was improved (25% released in 24 h) by precipitating the particles using aqueous sodium deoxycholate solution (DOC-modified PLA lamellae and lyophilising the lamellae-protein preparation after adsorption. Sustained immune responses were elicited in mice to a single sub-cutaneous injection of OVA adsorbed onto DOC-modified PLA lamellae. The level of antibodies induced and the pattern of response was similar to that induced by an alum-adsorbed OVA formulation. Normally boosting is required to obtain high levels of antibody when OVA is adsorbed on poly(DL-lactide co-glycolide) (PLG) microspheres. The lamellar forms of PLA may function as an efficient immunomodulator by effectively retaining adsorbed antigen and by activating immune cells due to their irregular shape. PLA lamellae have potential to stimulate enhanced immune responses to a variety of adsorbed antigens.

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.

Bile reflux into the stomach has been considered carcinogenic. Secondary bile acids, and in particular deoxycholic acid, have been shown to act experimentally as co-carcinogens in the colon and are increased in patients with colorectal adenocarcinoma. No information is available with respect to biliary bile acid composition in patients with gastric cancer. We studied biliary bile acid composition in 11 patients with gastric cancer and 23 healthy controls. Bile acids were measured using high-performance liquid chromatography. The site of gastric cancer was the antrum in 6 patients and body in 5. There were 6 intestinal-type and 5 diffuse adenocarcinomas. Only 2 patients had Helicobacter pylori infection. Deoxycholic acid constituted 24% +/- 2% of biliary bile acid in gastric cancer patients versus 22% +/- 2% in healthy controls (NS). Similarly, no differences were found between the two groups for all other bile acids. Deoxycholic acid constituted 23% +/- 3% of biliary bile acid (NS vs. controls) in patients with antral adenocarcinoma and 25% +/- 2% (NS vs. controls) in patients with intestinal-type gastric adenocarcinoma. Gastric adenocarcinoma is not associated with an increase in the more-toxic secondary bile acids, and deoxycholic acid in particular. This reduces the importance of bile acid composition as a promotor in gastric carcinogenesis.

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M(-1) min(-1). The pH-inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.

While UDP-glucuronosyltransferases (UGTs) are known to be expressed at high levels in human liver, relatively little is known about extrahepatic expression. In the present study, UGT2B family isoforms involved in the glucuronidation of steroid hormones and bile acids have been characterized in microsomes prepared from jejunum, ileum and colon from six human subjects. Glucuronidation of androsterone and testosterone was highly significant and increased from proximal to distal intestine. In contrast, hyodeoxycholic acid was glucuronidated at a low level in jejunum and ileum and activity was barely detectable in colon. No significant glucuronidation of lithocholic acid was found. Small phenols were glucuronidated with much lower activity than found in liver. High levels of UGT protein were detected with polyclonal anti-rat androsterone- and testosterone-UGT antibodies, whereas UGT2B4, a major hepatic hyodeoxycholic acid-specific UGT, was undetectable using a highly specific anti-human UGT2B4 antibody. Screening for RNA expression by RT-PCR confirmed the absence of UGT2B4 and UGT1A6 and showed expression of UGT2B7, a hepatic isoform shown to glucuronidate androsterone, in all intestinal segments. To our knowledge, the presence of functional androsterone and testosterone directed isoforms in human intestine is a novel finding which supports the idea that the intestinal tract functions as a steroid-metabolizing organ and plays a significant role in steroid hormone biotransformation.

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.

These studies used mice that were deficient in cholesterol 7alpha-hydroxylase to determine the effects of reduced bile acid synthesis on cholesterol homeostasis. In mice lacking this enzyme, bile acid synthesis was reduced from 8.3 to 3.4 micromol/day per 100 g body weight, the intestinal bile acid pool was decreased from 62.5 to 13.2 micromol/100 g bw, and the proportion of hyodeoxycholate, relative to cholate, in this pool was significantly increased. Associated with these changes, intestinal cholesterol absorption decreased from 37% to <1% while triacylglycerol absorption and animal weight gain remained essentially unaffected. The very low rate of cholesterol absorption could be corrected by feeding the mutant mice cholate, but not hyodeoxycholate. The reduction in sterol uptake across the intestine was associated with a 2-fold increase in cholesterol synthesis in the small bowel and liver and an increase in fecal neutral sterol excretion from 15.2 to 35.7 micromol/day per 100 g bw. The size of the cholesterol pools in the plasma, various organs and whole animal remained constant. Thus, under circumstances where the excretion of sterol as bile acids was markedly reduced, total cholesterol turnover actually increased from 164 to 239 mg/day per kg bw. This study demonstrates the complex interactions between bile acid and cholesterol metabolism and the dramatic effects of eliminating a single gene product; however, even though a major catabolic pathway was deleted, cholesterol balance across the animal was maintained.

We recently showed that feeding the cytoprotective bile acid ursodeoxycholic acid (UDCA) to rats resulted in significant reduction in polyps and especially cancers, both in number and size (D. L. Earnest et al., Cancer Res., 54: 5071-5074, 1994). Because fecal secondary bile acids [particularly deoxycholic acid (DCA)] are considered to promote formation of colon adenomas and cancer, we have now attempted to find a relationship between polyp reduction and fecal secondary bile acids after feeding UDCA to these rats. We examined the fecal bile acids in rats with polyps and compared them with fecal bile acids in control rats and also determined the bile acid composition in fecal aqueous phase, which is in direct contact with the colon epithelium and may be physiologically more active. Treatment with azoxymethane did not significantly alter fecal bile acid composition in the rats. Cholic acid feeding resulted in greatly increased proportions of DCA (82% of total bile acids versus 18% in control rats). On the other hand, UDCA feeding significantly reduced the proportion of fecal DCA (2% in control rats fed UDCA and 3% in rats also treated with azoxymethane). In control rats, 96% of the bile acids were present in the water-insoluble fraction and 4% in the water-soluble fraction. The major insoluble bile acids included DCA and hyodeoxycholic acid (73% of total bile acids). In contrast, the muricholic acids were concentrated in the soluble fraction (37%). When 0.4% UDCA was added to the diet, lithocholic acid increased in the insoluble fraction (40 versus 1%), but the hydrophilic UDCA and muricholic acids were enriched in the water-soluble fraction (37 and 43%, respectively). Thus, the hydrophobic bile acids were distributed predominantly in the water-insoluble fraction, whereas the hydrophilic bile acids were distributed preferentially in the water-soluble fraction. These data suggest that UDCA may prevent colon tumors and polyps by countering the toxic effect of DCA and enhancing the possible cytoprotective effects of UDCA and muricholic acids in the water-soluble fraction in the feces of rat.

An aminopeptidase activity capable of hydrolyzing different aminomethylcoumarin amino acids, but mainly leucine-7-amino-4-methylcoumarin (Leu-NHMc), was detected in deoxycholic soluble extracts from adult Fasciola hepatica. The enzyme (EC 3.4.11.1) was partially purified by gel filtration and EAH-Sepharose affinity chromatography using bestatin as a ligand. Results obtained by gel filtration, direct fluorogenic substrate analysis in polyacrylamide gel, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that in a native form the enzyme might be aggregated as a high molecular weight complex. By affinity chromatography on concanavalin A Sepharose, the enzyme did not bond to the column showing that it lacks mannose residues. The F. hepatica aminopeptidase was characterized as a metalloproteinase based on its activation by Mn2+ and Mg2+, and its inhibition by EDTA, 1,10-phenanthroline, and bestatin. It has an optimal activity at a pH between 8.0 and 8.5. Histochemical localization revealed strong leucine naphthylamidase activity at the cells lining the gut epithelium of the parasite.

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.

Iron(II) ions react with small aggregates of cholate, glycocholate, chenodeoxycholate, and deoxycholate to form soluble and colloidal compounds. Taurocholate under conditions used does not react with the Fe2+ ion. Small aggregates of dihydroxy bile salts (predominating in the premicellar region, at concentrations of the bile salt above 1 mmol dm-3) have a larger affinity for Fe2+ compared to those formed from cholate anions. In their interactions with small aggregates of cholate anions, the Fe2+ ion shows an affinity comparable to that of Cu2+ and Cd2+ and somewhat larger than that of Zn2+. Small aggregates of cholate show a higher ability to mask Fe2+ than those of taurocholate and glycocholate. Interaction of glycocholic acid anions with Fe2+ ions is sufficient to prevent iron(II) precipitation.

A method based on coupling of the techniques of radioscanning a TLC plate and densitometry has been developed for the determination of pool sizes and fractional turnover rate of bile acids in man after intraduodenal administration of 14C-labelled acid. The validity of the method has been checked by comparison of the results obtained with those of an enzymatic spectrophotometric analysis, and a measurement of the radioactivity by liquid scintillation counting, after elution of the separated bile acid from a TLC plate. Advantages of the proposed method over the previous one include a reduced number of manipulations, the possibility of automation, a better reproducibility, and the possibility of elaborating the radiometric data obtained for the primary bile acid for better characterising its metabolism inside the enterohepatic circulation.

Objective: To compare the effect of short term feeding of ursocholic acid, a hydrophilic bile acid, as the unconjugated acid and the taurine conjugate, on clinical and biochemical features and bile acid metabolism with that of ursodeoxycholic acid in four patients with primary biliary cirrhosis., Methods: Four patients with stage II primary biliary cirrhosis were studied. Two were fed ursocholic acid (900 mg/day), and two were given tauroursocholate (900 mg/day) in three divided doses. After 1 month, all patients were given 900 mg/day of ursodeoxycholic acid. Fasting serum, bile, and 24-hour urine levels were measured before and at the end of ursocholic acid and tauroursocholate feeding and after 1 month of ursodeoxycholic acid feeding. Clinical and biochemical symptoms were measured by routine hospital methods, and bile acids were measured by gas-liquid chromatography., Results: One month of ursocholic acid or tauroursocholate feeding did not improve clinical or biochemical findings in any patient. Approximately 21-25% ursocholic acid was present in the serum and bile, with substantial metabolism to deoxycholic acid. Increased ursocholic acid was excreted in the urine. In comparison, ursodeoxycholic acid improved biochemical parameters and was 45-65% enriched in the serum and bile., Conclusion: Ursocholic acid as the free bile acid or as taurine conjugate, although more hydrophilic, is poorly enriched in serum and bile and is ineffective in patients with primary biliary cirrhosis.

Amphotericin B lipid complex (ABLC) shows reduced toxicity relative to that of amphotericin B deoxycholate (AmB-d) while maintaining antifungal activity. Rat blood or plasma was spiked with ABLC in vitro. Released amphotericin B was separated from the parent material by centrifugation. At early times (0 to 15 min) most (approximately 90%) of the amphotericin B was complexed. The amount of released amphotericin B increased gradually in a time- and temperature-dependent fashion. The released amphotericin B was associated with plasma lipoprotein and nonlipoprotein proteins. The area under the concentration-time curve from 0 to 24 h for total amphotericin B in whole blood of rats given a single intravenous bolus dose of 1 mg of ABLC per kg of body weight was fourfold lower than that in rats given 1 mg of AmB-d per kg. The complexed amphotericin B was rapidly removed from the circulation and was distributed to the tissues in these rats. Other rats were treated intravenously with ABLC (10 mg/kg/day) or AmB-d (0.5 mg/kg/day) daily for 15 days. Blood was collected at 15 and 180 min after administration of the last dose. The total levels of amphotericin B in the blood of the group given ABLC were about three to five times those in the group given AmB-d, and the concentration of released, protein-bound amphotericin B in the plasma of the group given ABLC was about one to two times that observed for the group given AmB-d, despite the 20-fold difference in dose. The relative protein distribution of amphotericin B in plasma was similar after ABLC or AmB-d administration under these steady-state conditions in vivo. The rapid uptake of complexed amphotericin B by tissues and the very low levels of circulating protein-bound amphotericin B in plasma after the administration of ABLC may explain, in part, the reduced toxicity and enhanced therapeutic index of this preparation.

The primum movens in cholesterol gallstone formation is hepatic cholesterol hypersecretion and chronic supersaturation of bile. From this event a cascade of contributing factors can be differentiated: (i) Motility defects with impaired gallbladder contractility and gallbladder stasis, but also with small and large intestinal hypomotility. (ii) Multiple biochemical defects in gallbladder bile with increased biliary proteins, increased deoxycholic acid and rapid crystallization of biliary cholesterol from supersaturated unstable vesicles. There is considerable evidence that slow intestinal and colonic transit can increase the deoxycholic acid pool size and biliary cholesterol saturation. Changes in intestinal transit influence the anaerobic bacterial enzymatic biotransformation of conjugated cholate to more hydrophobic deoxycholate. This leads to biliary cholesterol hypersecretion and gallstone formation. Prokinetic drugs or administration of lactulose or fiber products like bran can change the slow intestinal transit favourably with subsequent reduction in deoxycholic acid formation and cholesterol saturation of bile. Whether these applications are indeed of value in the long-term prevention of gallstone disease, however, is doubtful, since fiber-rich diet in prevention of gallstone recurrence after complete gallstone dissolution was not successful.

Secondary bile acids (BA) have been shown to be involved as a promoting agent in the adenoma-carcinoma sequence of colorectal cancer. In previous studies, fermentation of starch has been shown to inhibit the degradation of primary to secondary BA by the colonic microflora. This study was designed to investigate BA metabolism in continuous cultures of mixed fecal bacteria to get further insights into the mechanisms of this inhibition. Fermentation vessels were fed with media containing cholic (0.6 g/l) and chenodeoxycholic acid (0.4 g/l). Cultures were either starch-free or enriched with starch (10 g/l). pH was controlled and adjusted to 7.0 or 6.0. Total culture duration was 28 days and concentrations of BA, short-chain fatty acids (SCFA), and starch were measured periodically. At pH 6, significantly more primary BA remained in the media and less secondary BA were produced. Total BA concentrations were lower at pH7. SCFA concentrations were higher in the vessels supplemented with starch. Starch was completely fermented and not present in significant amounts in any fermentation vial after the first week. These data indicate that bacterial breakdown of primary to secondary BA is inhibited when starch is simultaneously fermented. This effect can be explained by the reduction of pH resulting from SCFA production. Considering these findings, resistant starch which escapes assimilation in the small bowel may be a protective factor against colorectal cancer.

Forty-four strains of a phenotypically unique Campylobacter were isolated from the faeces of 26 of 45 cows in a single herd. Isolation involved enrichment and membrane filtration onto blood agar or plating onto cefoperazone amphotericin teicoplanin agar. The strains exhibited phenotypic characteristics typical for Campylobacter species. However, they were unusual in that they produced urease and copious H2S in triple sugar iron (TSI) medium, but did not produce catalase. They did not grow aerobically. None of the strains grew on modified cefoperazone charcoal deoxycholate agar (mCCDA). Macrorestriction profiles of chromosomal DNA were prepared for 15 strains using pulsed-field gel electrophoresis (PFGE). Twelve of 15 profiles were identical and all appeared to be closely related. These catalase-negative, urease-positive campylobacters (CNUPC) represent a group not previously reported. Their sensitivity to antibiotics normally used in selective media for campylobacters might explain why they have not previously been encountered. Their ecological significance and importance with respect to human and animal disease remain to be assessed.

In 41 healthy human-milk-fed preterm infants the preprandial total bile acid (BA) concentrations in serum and duodenal juice were simultaneous measured during the first 60 days of life. The infants were subdivided into four groups according to their gestational age: 6 infants with a gestational age of 27 and 28 weeks, 7 infants with a gestational age of 29 and 30 weeks, 21 infants with a gestational age of 31 and 32 weeks and 7 infants with a gestational age of 33 and 34 weeks. The BA levels were enzymatically determined using 3-alpha-hydroxysteroid dehydrogenase. In the duodenal juice, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid and lithocholic acid were separately quantified by thin-layer chromatography. During the first month of life, the serum BA concentrations increased significantly with postnatal age (p < 0.01) but remained nearly constant during the second month of life. In the duodenal aspirates, the BA concentrations increased continuously up to the end of the observations period (p < 0.001). In the duodenal aspirates, the CA/CDCA ratio was high immediately after birth and decreased significantly with increasing postnatal age (p < 0.001). During the first weeks of life, the BA levels were preferentially conjugated with taurine, but in spite of the taurine-rich diet during the whole observation period the taurine/glycine ratio decreased with postnatal age (p < 0.001). In all samples of duodenal juice, the sum of primary BA was > 98% of total 3-alpha-hydroxy-BA. These data indicate that the establishment of an intestinal microbial flora necessary for intestinal BA transformation and the development of the enterohepatic BA circulation lasts some months of postnatal life. The serum BA concentration reflects hepatic synthesis, intestinal absorption, renal excretion and hepatocellular transport into bile in a very complex way which may limit the diagnostic value of serum BA during this time. Additionally, a duodenal BA concentration below 4 mmol/l, as found in this study during the first 2 weeks of life, may be of clinical importance due to its possible effects on fat absorption.

Background & Aims: Excessive deoxycholic acid (DCA) in the bile acid pool with cholesterol supersaturation of bile is prevalent in patients with cholesterol gallstones (CGs). This study examined whether this is caused by enhanced conversion of cholic acid (CA) to DCA by intestinal bacteria., Methods: Ten patients with CGs with DCA excess (DCA/CA pool ratio, > 1.5) and 10 patients with low DCA (ratio, < 1.0) were compared for CA and DCA kinetics, ileal absorption of 75-Se-homotaurocholic acid (75-SeHCAT), and CA-7 alpha-dehydroxylation activity of the fecal microflora; the effects of ampicillin treatment on DCA excess were studied in 7 patients., Results: Patients with DCA excess and low DCA differed (P < 0.01) in the pool size of CA (mean, 5.8 vs. 34) and DCA (28 vs. 11 mumol/kg) and DCA input (8.8 vs. 3.5 mumol.kg-1.day-1. Whereas 75-SeHCAT excretion was similar, CA-7 alpha-dehydroxylation activity and levels of fecal 7 alpha-dehydroxylation bacteria were 3-fold and 1000-fold higher (P < 0.01) in patients with DCA excess, respectively. Ampicillin treatment decreased (P < 0.02) CA-7 alpha-dehydroxylation activity and DCA pool size, expanded the CA pool to normal size, and lowered cholesterol saturation of bile., Conclusions: Increased CA-7 alpha-dehydroxylation activity of the intestinal microflora may be an important factor for CG formation or growth in these patients.

In isolated hamster hepatocytes, the Ca2+ ionophore A-23187 immediately decreased the uptake rate of taurocholic acid (TCA) by 60-70%, whereas it slowly inhibited that of ursodeoxycholic acid (UDCA) by a maximum of 35-45%, with an inhibition constant (Ki) of 0.36 and 1.93 microM, respectively. In contrast to ionomycin, which mimicked the effect of A-23187, vasopressin inhibited the bile acid uptake rate by 40 and 45%, respectively, only after a 5- to 10-min preincubation. The Na(+)-dependent bile acid transport was exclusively inhibited by these agents, and this inhibition was independent of extracellular Ca2+. However, intracellular Ca2+ depletion with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid resulted in 40-50% inhibition of the uptake rate of both bile acids. The exogenous protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), but not the nonactive 4 alpha-phorbol, significantly inhibited TCA uptake rate. Although both A-23187 and ionomycin immediately increased and decreased the cellular Na+ and K+ concentration, respectively, neither vasopressin nor PMA had a significant effect on the cellular concentration of these cations, even after a 10-min incubation. Furthermore, the effect of A-23187 and ionomycin on TCA uptake and Na+ flux, respectively, disappeared after a 40-min preincubation, and additional ionophore remained without effect. However, after a 40-min incubation with A-23187, PMA was still able to inhibit TCA uptake. Therefore, A-23187 and ionomycin transiently inhibited Na(+)-dependent uptake of both TCA and UDCA, in part because of transient alteration of the cellular Na+ and K+ concentration. Vasopressin and PMA inhibited Na(+)-dependent bile acid uptake, at least in part, through protein kinase C activation.

Unlabelled: A structure-activity relationship for bile acid (BA) intestinal absorption is known to exist. To better understand the BA structural requirements for optimal BA intestinal absorption, rabbit ileal perfusion studies were performed. Unconjugated BA: Ursodeoxycholic (UDCA) and ursocholic acid (UCA) with methyl (6MUDCA and 6MUCA) or fluoro group (6FUDCA and 6FUCA) in the 6 position and UCA with a methyl group in 23 position (23MUCA) were compared with unconjugated UDCA, UCA, deoxycholic (DCA), chenodeoxycholic (CDCA), hyocholic (HCA), and hyodeoxycholic (HDCA) acid. BA lipophilicity was evaluated by their octanol-water partition coefficient. Conjugated BA: Taurine-conjugated UDCA and UCA with a methyl group in the 23 position (T23MUDCA and T23MUCA) were compared with the corresponding taurine-conjugated natural analogs. An analog of glycine-conjugated UDCA with the C24 amide bond replaced by a -CO-CH2- in the 24 position (24PUDCA) was studied and results were compared with the natural form (GUDCA). Unconjugated BA absorption was dose dependent (i.e., passive) and followed their lipophilicity: DCA > 6MUDCA > CDCA > HDCA > UDCA > HCA > 6FUDCA > 6MUCA > 6FUCA > UCA. Conjugated BA absorption was active, and Vmax was in the following order: TCA > TUDCA > TUCA > T23MUCA > T23MUDCA > 24PUDCA > GUDCA. 24PUDCA transport was also active and higher than GUDCA., Conclusion: Passive transport is dependent on BA lipophilicity. Conjugated BAs are actively transported, and the presence of a 23-C methyl group does not improve transport when compared with the natural analogs. The substitution of the C24 amide bond with a -CO-CH2-still affords interaction of the BA with the intestinal transport carrier.

Use of calcium supplements has increased dramatically in recent years yet little is known about the effect of calcium supplementation on colon physiology. We supplemented 22 individuals with a history of resected adenocarcinoma of the colon, but currently free of cancer, with 2000 or 3000 mg calcium for 16 wk. The effects of supplementation on duodenal bile acids and important fecal characteristics including total fecal output, wet and dry weight, pH, bile acids (in solids and in fecal water), and concentrations and total excretion of calcium, magnesium, phosphates (organic and inorganic), unesterified fatty acids and total fat were determined. Calcium supplementation significantly decreased the proportion of water in the stool (P = 0.03), doubled fecal excretion of calcium (P = 0.006), and increased excretion of organic phosphate (P = 0.035) but not magnesium. Calcium supplementation significantly decreased the proportion of chenodeoxycholic acid in bile (P = 0.007) and decreased the ratio of lithocholate to deoxycholate in feces (P = 0.06). The concentration of primary bile acids in fecal water decreased after 16 wk Ca supplementation. Together with other reports of a "healthier" bile acid profile with respect to colon cancer when changes such as those observed in this study were achieved, these results suggest a protective effect of calcium supplementation against this disease.

The efficiency of bile acid conjugation before and during therapy with 600 mg/day of ursodeoxycholic acid was measured in seven adult patients with early chronic cholestatic liver disease (6 with primary biliary cirrhosis; 1 with primary sclerosing cholangitis). Duodenal bile samples were obtained by aspiration and the proportion of unconjugated bile acids was determined using lipophilic anion exchange chromatography to separate bile acid classes, followed by analysis of individual bile acids by gas chromatography-mass spectrometry. The proportion of conjugated bile acids was determined by high-performance liquid chromatography. Use of a (99m)Tc-HIDA recovery marker permitted the absolute mass of unconjugated bile acids in the gallbladder to be calculated. Unconjugated bile acids comprised 0.4% of total biliary bile acids before and 0.2% during ursodeoxycholic acid therapy, indicating highly efficient conjugation of bile acids. During therapy, percentage unconjugated ursodeoxycholic acid significantly increased from (mean +/- S.D.) 13 +/- 13% to 54 +/- 12%; P < 0.002. When the unconjugated and conjugated fractions of bile acids were compared, there was an enrichment in unconjugated fraction for cholic acid and ursodeoxycholic acid and a depletion for chenodeoxycholic acid both in basal condition and during ursodeoxycholic acid therapy, suggesting that hydrophilic bile acids were conjugated less efficiently. During therapy, the conjugation efficiency significantly increased for cholic acid and ursodeoxycholic acid. The pretreatment mass of total unconjugated bile acids in the gallbladder was (mean +/- S.D.) 4.4 +/- 3.2 mumol, and was not significantly changed by ursodeoxycholic acid therapy (6.2 +/- 3.5 mumol). However, ursodeoxycholic acid therapy caused a significant increase in the mass of unconjugated ursodeoxycholic acid. It is concluded that endogenous bile acids and exogenous ursodeoxycholic acid when given at the usual dose are efficiently conjugated in patients with early cholestatic liver disease. Despite showing increased biliary unconjugated ursodeoxycholic acid during its oral administration, our data do not lend support to the occurrence of hypercholeresis due to cholehepatic shunting of bile acids.

Bile acids labeled with 3H on the sterol nucleus lose a substantial fraction of label during enterohepatic cycling and conversion to secondary bile acids. We tested the isotopic stability of a side-chain 3H label, [22,23-3H]cholic acid in humans. The 3H-labeled compound was administered simultaneously with [24-14C]cholic acid to four healthy volunteers. Duodenal bile was collected daily for 5 days after isotope administration to determine the ratio of 3H/14C in bile acids. Urine was collected to determine loss of radioactivity by this route. Cholic acid and deoxycholic acid were isolated from biliary bile acids by thin-layer chromatography after deconjugation with cholylglycine hydrolase. The ratio of 3H/14C in cholic acid and deoxycholic acid remained constant and identical to that of the administered mixture in all subjects, indicating stability of the 3H label during enterohepatic cycling. Cumulative loss of 3H in urine averaged only 1.2% of administered dose and was identical to loss of 14C (average 1.3%) indicating little if any transfer of 3H from bile acid to body water. Deconjugation of biliary bile acids by alkaline hydrolysis resulted in 15-20% loss of 3H label, consistent with known base-catalyzed exchange of alpha-carbon protons on carboxylic acids. We conclude that [22,23-3H]cholic acid is a biologically stable, and therefore reliable, isotopic tracer of cholic acid in humans during enterohepatic cycling including conversion to deoxycholic acid, provided deconjugation is performed enzymatically. Because the 22,23-3H label can be inserted into most C24 bile acids, it appears the best way to tag 3H-labeled bile acids for metabolic studies.

Background & Aims: A direct comparison of jejunal and ileal absorption rates of bile acids has not been reported. The aim of this study was to compare the relative transport rates of different bile acids in the jejunum and ileum., Methods: Jejunal and ileal rabbit intestinal segments were separately perfused with bile acid solutions, and dose-response curves were obtained for taurocholate, ursodeoxycholate, chenodeoxycholate, deoxycholate, and their glycoconjugates. Membrane fluidity and bile acid transport were assessed in brush border membrane vesicles., Results: Taurocholate showed active transport in the ileum and no transport in the jejunum. Unconjugated bile acids showed passive diffusion in the two tracts, whereas glycoconjugated bile acids showed both components of transport in the ileum and passive diffusion in the jejunum (lower in the latter). A higher membrane fluidity and lower cholesterol-to-phospholipid ratio were found in the jejunum. Ursodeoxycholate reduced bile acid uptake into membrane vesicles from both ileum and jejunum., Conclusions: Active transport is limited to the ileum. Passive diffusion is higher through a less fluid membrane with a higher cholesterol-to-phospholipid ratio in the ileum than in the jejunum. Ursodeoxycholate inhibition may be at the level of a facilitated, sodium-independent diffusion in the jejunum.

Experimental dietary studies of human colorectal carcinogenesis are usually based on the AIN-76A diet, which is dissimilar to human food in source, preparation, and content. The aims of this study were to examine the feasibility of preparing and feeding rats the diet of a specific human population at risk for colorectal neoplasia and to determine whether changes in the colonic morphology and metabolic contents would differ from those resulting from a standard rat diet. The mean daily food intake composition of a previously evaluated adenoma patient case-control study was used for the "human adenoma" (HA) experimental diet. Foods were prepared as for usual human consumption and processed by dehydration to the physical characteristics of an animal diet. Sixty-four female Sprague-Dawley rats were randomized and fed ad libitum the HA or the AIN-76A diet. Every eight weeks, eight rats from each group were sacrificed, and the colons and contents were examined. Analysis of the prepared food showed no significant deleterious changes; food intake and weight gain were similar in both groups. Compared with the controls, the colonic contents of rats fed the HA diet contained significantly less calcium, concentrations of neutral sterols, total lipids, and cholic and deoxycholic acids were increased, and there were no colonic histological changes other than significant epithelial hyperproliferation. This initial study demonstrated that the HA diet can be successfully processed for feeding to experimental animals and is acceptable and adequate for growth but induces significant metabolic and hyperproliferative changes in the rat colon. This dietary model may be useful for studies of human food, narrowing the gap between animal experimentation and human nutritional research.

Hepatitis C virus (HCV) in highly infectious sera has been shown to be predominantly associated with low-density lipoproteins. To determine whether the association is specific to low-density lipoproteins (LDL) or very low-density lipoproteins (VLDL), we fractionated HCV-containing plasma by a column chromatographic procedure known to separate these classes. Hepatitis C virus RNA detected by polymerase chain reaction (PCR) was associated primarily with the very low-density (VLDL) fraction. However, it could not be ruled out that virus-associated LDL may have eluted with this fraction. Hepatitis C virus virions isolated from sera having sufficient titre for visualization by electron microscopy are generally coated with antiviral antibodies, therefore we utilized the lipid association to isolate antibody-free virions. Very low-density lipoproteins were isolated by ultracentrifugal flotation and then treated with deoxycholate to release the virions. These were then isolated in a highly purified form by centrifugation in a sucrose gradient. The 1.10-1.11 g ml-1 region of the gradients contained 60-70 nm particles. Particles with similar surface structure but having a diameter of only 30-40 nm constituted about 30% of the total. The latter may represent defective interfering particles. The identity of both small and large particles with HCV virions and associated particles was confirmed by their trapping on grids by an anti-HCV E2 monoclonal antibody, and by their aggregation by rabbit antiserum to an amino-terminal peptide of E1. Thus, both E1 and E2 epitopes are displayed on the surface of intact HCV virions.