BACKGROUND:Acromegalic patients have slow colonic transit, increased rates of deoxycholic acid formation, and an increased prevalence of cholesterol gall stones, especially during long term octreotide treatment. However, the effects of this prolonged large bowel transit time on the numbers of faecal anaerobes and the activities of the enzyme systems which biotransform conjugated cholic acid into unconjugated deoxycholic acid (cholylglycine hydrolase and 7alpha-dehydroxylase) are unknown. METHODS:Therefore, in 10 non-acromegalic controls, 11 acromegalic patients not treated with octreotide, and 11 acromegalics on long term (8-48 months) octreotide (100-200 mug three times daily subcutaneously), we measured large bowel transit time and, in freshly voided faeces, the activities of the two bile acid metabolising enzymes, and related the results to the proportion of deoxycholic acid in fasting serum. Moreover, in patients with acromegaly, we measured quantitative bacteriology in faeces. RESULTS:Mean large bowel transit time in acromegalics not treated with octreotide (35 (SEM 6.5) hours) was 66% longer than that in non-acromegalic controls (21 (3.1) hours; NS) and became further prolonged during octreotide treatment (48 (6.6) hours; p<0.001). These octreotide induced changes in transit were associated, in acromegalic patients, with more total (15.0 (2.5) v 6.3 (1.3)x10(9) colony forming units (cfu)/g; p<0.05) and Gram positive (6.3 (2.3) v 3.2 (1.0)x10(9) cfu/g; p<0.05) faecal anaerobes. Mean faecal cholylglycine hydrolase activity in the long term octreotide group (22.0 (6.0)x10(-2) U/mg protein) was 138% greater than that in non-acromegalic controls (12.0 (6.0)x10(-2); p<0.01). Similarly, mean 7alpha-dehydroxylase activity in octreotide treated acromegalics (11.1 (1.18)x10(-4) U/mg protein) was 78% greater than that in patients not receiving long term octreotide (6.3 (0.5)x10(-4); p<0.001). The mean proportion of deoxycholic acid in fasting serum also increased from 18.0 (2.88)% in the untreated group to 29.6 (2.3)% during long term octreotide (p<0.05). There were significant linear relationships between large bowel transit time and: (i) faecal 7alpha-dehydroxylase activity; and (ii) the proportion of deoxycholic acid in fasting serum and between 7alpha-dehydroxylase activity and the proportion of deoxycholic acid in serum.Summary/interpretation: These data suggest that increased deoxycholic acid formation seen in acromegalics during octreotide treatment is due not only to the greater numbers of faecal anaerobes but also to increased activity of the rate limiting enzyme pathway (7alpha-dehydroxylation) converting cholic acid to deoxycholic acid.

BACKGROUND:Inflammatory bowel disease is a risk factor for gall-bladder stones, but there is controversy about the composition of these stones and whether such patients develop lithogenic bile.METHODS:In 54 gallstone-free inflammatory bowel disease patients and 13 non-inflammatory bowel disease patients with cholesterol-rich gallstones, we measured the biliary cholesterol saturation indices, nucleation times and bilirubin concentrations, and determined the bile acid composition and molecular species of phosphatidylcholine, in gall-bladder bile.RESULTS:Patients with Crohn's colitis or ulcerative colitis had less saturated bile (mean cholesterol saturation index, 0.9) and longer nucleation times (median, 21 days) than those with ileal Crohn's disease (1.5; 14 days) or those who had undergone colectomy (1.6; 5 days). In patients with ileal Crohn's disease, the mean biliary bilirubin concentration was two- to three-fold higher than that in the other groups, and was associated with a decrease in the percentage of biliary deoxycholate and an increase in the percentage of ursodeoxycholate, compared with disease controls, but phosphatidylcholine species were similar.CONCLUSIONS:Patients with small bowel Crohn's disease, or who have undergone colonic resection, have supersaturated bile and an increased risk of cholesterol gallstone formation. In patients with ileal disease, the presence of high biliary bilirubin concentrations and low percentage of deoxycholic acid may also favour the formation of mixed, pigment-rich, gallstones.

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.

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.

BACKGROUND:Octreotide inhibits gall bladder emptying and prolongs intestinal transit. This leads to increases in the proportion of deoxycholic acid in, and cholesterol saturation of, gall bladder bile, factors that contribute to the pathogenesis of octreotide induced gall stones.AIMS:To see if an intestinal prokinetic, cisapride, could overcome these adverse effects of octreotide and if so, be considered as a candidate prophylactic drug for preventing iatrogenic gall bladder stones.METHODS:A randomised, double blind, placebo controlled, crossover design was used to examine the effects of cisapride (10 mg four times daily) on gall bladder emptying, mouth to caecum and large bowel transit times, and the proportions of deoxycholic acid and other bile acids, in fasting serum from: (i) control subjects (n=6), (ii) acromegalic patients not treated with octreotide (n=6), (iii) acromegalics on long term octreotide (n=8), and (iv) patients with constipation (n=8).RESULTS:Cisapride had no prokinetic effect on the gall bladder. In fact, it significantly increased both fasting and postprandial gall bladder volumes. However, it shortened mouth to caecum (from 176 (13) to 113 (11) minutes; p<0.001) and large bowel (from 50 (3.0) to 31 (3.4) h; p<0.001) transit times. It also reduced the proportion of deoxycholic acid in serum from 26 (2.3) to 15 (1.8)% (p<0.001), with a reciprocal increase in the proportion of cholic acid from 40 (3.5) to 51 (3.8)% (p<0.01). There were significant linear relationships between large bowel transit time and the proportions of deoxycholic acid (r=0.81; p<0.001) and cholic acid (r=-0.53; p<0.001) in fasting serum. INTERPRETATION/SUMMARY: Cisapride failed to overcome the adverse effects of octreotide on gall bladder emptying but it countered octreotide induced prolongation of small and large bowel transit. Therefore, if changes in intestinal transit contribute to the development of octreotide induced gall bladder stones, enterokinetics such as cisapride may prevent their formation.

Background & Aims: Prolonged large bowel transit, and an increase in the proportion of deoxycholic acid (DCA), have been implicated in the pathogenesis of cholesterol gallstones-including those developing in acromegalics treated with octreotide. However, there are few data on the effects of intestinal transit on bile acid kinetics. Methods: We therefore measured the kinetics of DCA and cholic acid (CA) using stable isotopes, serum sampling, and mass spectrometry. The results were related to mouth-to-caecum (MCTT) and large bowel transit times (LBTTs) in 4 groups of 8 individuals: (1) non-acromegalic controls, (2) acromegalics untreated with octreotide, (3) acromegalics on long-term octreotide, and (4) patients with constipation. Paired, before and during octreotide, studies were performed in 5 acromegalics. Results: In the unpaired and paired studies, octreotide significantly prolonged MCTT and LBTT. In the paired studies, the octreotide-induced prolongation of LBTT caused an increase in the DCA input rate (6.4 +/- 2.8 to 12 +/- 2.6 mu mol . kg . d, P < 0.05) and pool size (18 +/- 12 to 40 +/- 13 mu mol/kg, P < 0.05), and a decrease in CA pool size (45 +/- 15 to 25 +/- 11 mu mol/kg, P < 0.05). Furthermore, during octreotide treatment, the mean conversion of C-13-CA to C-13-DCA (micromoles) was greater (P < 0.05) on study days 3, 4, and 5. There were also positive linear relationships between LBTT and DCA input rate (r = 0.78), pool size (r = 0.82, P < 0.001), and a weak (r = -0.49) negative linear relationship between LBTT and CA pool size (P < 0.01). Conclusions: These data support the hypothesis that, by increasing DCA formation and absorption, prolongation of large bowel transit is a pathogenic factor in the formation of octreotide-induced gallstones.

BACKGROUND Deoxycholic acid (DCA), implicated in the pathogenesis of gall stones and colorectal cancer, is mainly formed by bacterial deconjugation (cholylglycine hydrolase (CGH)) and 7α-dehydroxylation (7α-dehydroxylase (7α-DH)) of conjugated cholic acid (CA) in the caecum/proximal colon. Despite this, most previous studies of CGH and 7α-DH have been in faeces rather than in caecal contents. In bacteria, CA increases 7α-DH activity by substrate-enzyme induction but little is known about CA concentrations or CA/7α-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 7α-DH, (ii) measured 7α-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) 7α-DH activity and CA concentration in caecal samples (evidence of substrate-enzyme induction), and (v) 7α-DH and per cent DCA in serum. RESULTS Although mean CGH activity in the proximal colon (18.3 (SEM 4.40) ×10−2 U/mg protein) was comparable with that in “faeces” (16.0 (4.10) ×10− 2 U/mg protein) , mean 7α-DH in the caecum (8.54 (1.08) ×10-4 U/mg protein) was higher (p<0.05) than that in the left colon (5.72 (0.85) ×10-4 U/mg protein). At both sites, 7α-DH was significantly greater in the “high” than in the “low” serum DCA subgroups. CA concentrations in the right colon (0.94 (0.08) μmol/ml) were higher than those in the left (0.09 (0.03) μmol/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 7α-DH and CA concentration. At both sites, 7α-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 7α-DH influence per cent DCA in serum (and, by inference, in bile), and (iii) show that the substrate CA induces the enzyme 7α-DH in the caecum.

BACKGROUND & AIMS Many patients with cholesterol gallbladder stones (GBS) have a high percentage of deoxycholic acid (DCA) in gallbladder bile (all of which are in the conjugated form), probably as a result of prolonged large bowel transit times (LBTT). However, whether the prolonged LBTT increases DCA formation, solubilization, or absorption (or all 3) is not known. METHODS In 40 subjects (20 with GBS; age range, 24-74 years), we measured LBTT using radiopaque markers, and intestinal luminal pH by radiotelemetry. We also measured quantitative anaerobic bacteriology and the activities of 2 bile acid-metabolizing enzymes in fresh cecal aspirates obtained during clinically indicated unprepared colonoscopy, and related these results to the percentage of DCA in fasting serum measured by gas chromatography-mass spectrometry. RESULTS Compared with controls, GBS patients had longer LBTT (mean 23.1 +/- SEM 2.8 h vs. 36.5 +/- 3.3 h; P < 0.01); more total (2.7 +/- 0.6 x 10(9) vs. 5.9 +/- 1.5 x 10(9) cfu/mL) and Gram-positive (9.5 +/- 3.1 x 10(8) vs. 18.0 +/- 4.1 x 10(8) cfu/mL; P < 0.05) anaerobes; and greater 7alpha-dehydroxylating (7alpha-DH) activity (3.39 +/- 0.59 vs. 10.37 +/- 1.15 x 10(-4) U/mg protein) in the cecal aspirates. They also had higher intracolonic pH values (P < 0.02) and increased percentages of DCA in fasting serum (13.4% +/- 1.52% vs. 21.8% +/- 2. 19%; P < 0.005). Results of univariate and multivariate analyses confirmed that LBTT was critical in determining the percentage of DCA in serum and showed that 7alpha-DH activity and apparent distal colonic pH were also significant independent variables. CONCLUSIONS Slow colonic transit (more time), increased Gram-positive anaerobes (more bacteria), and greater 7alpha-DH activity (more enzyme) favor enhanced DCA formation; transit-induced increases in distal colonic luminal pH favor enhanced DCA solubilization/bioavailability; and increases in LBTT (more time) again favor DCA absorption.

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.

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 μM 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.

BACKGROUND:Although the prevalence of symptomatic gallbladder stone disease in Ghana increased almost four-fold between 1966 and 1991, little is known about the composition of and aetiopathogenesis of these stones, nor about their suitability for non-surgical (dissolution) treatments. METHODS:To study this, gallstones from 67 out of 90 patients coming to cholecystectomy were retrieved and, based on their external appearance, classified provisionally as cholesterol(chol) (n = 8), black pigment (n = 28) and brown pigment (n = 31) stones. The gallstones were then homogenised, their cholesterol(chol) content measured chemically and the stones re-classified as cholesterol-poor (< 10% chol by weight), intermediate (10-75% chol) and cholesterol-rich (> 75% chol). The relationship between the initial and the definitive classifications was then examined and the biliary bacteriology (carried out on fresh samples of gallbladder(GB) bile obtained by fine needle aspiration) on gallstone composition, analysed. RESULTS:The external appearance correctly predicted stone composition in the 28 thought, initially, to have black pigment stones (all of whom had stones containing < 10% chol by weight, on chemical analysis) and the eight believed, originally to have "cholesterol" stones (all of whom had stones with > 75% chol) but it proved unreliable in the 31 considered, at the time of surgery, to have brown pigment stones (mean chol content 58+ SEM 35%; range 0-98%. By chemical analysis, more than half the patients 35 of 67 or 52% had cholesterol-poor stones, nine (13% of the total) had intermediate stones, while 23 (34%) had cholesterol-rich stones. Cholesterol-rich gallstones were also more frequent in women than in men (p < 0.03). Only nine of 43 patients (21%) whose GB bile was aspirated, had positive bacterial cultures. There was no obvious difference in stone composition between those with positive, and those with negative, cultures. CONCLUSION:Since the majority of Ghanaian patients with cholecystolithiasis have gallbladder stones with < 75% chol by weight, when active treatment is indicated surgery is more appropriate than dissolution therapy. However contrary to common belief, cholesterol-rich gallstones do occur in West Africa: 34% of the present series had stones with > 70% chol by weight.

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 7α-dehydroxylation in the intestines. The levels and activities of bile acid 7α-dehydroxylating bacteria have been reported to be increased in gallstone patients. The aim of the current study was to isolate 7α-dehydroxylating bacteria from gallstone patients and determine if these individuals are colonized by similar bacterial species.