Current treatments of primary sclerosing cholangitis.

Primary Sclerosing Cholangitis (PSC) is a chronic cholestatic disease characterized by hepatic inflammation and obliterative fibrosis, resulting in both intra- and extra-hepatic bile duct strictures. End-stage liver disease and bile duct carcinoma represent frequent complications. Incidence and prevalence of PSC in USA have been recently estimated as 0.9 per 100,000 person-years, and 1-6 per 100,000 person-years, respectively. Major diagnostic criteria include the presence of multifocal strictures, beadings of bile ducts, and compatible biochemical profile, once excluded secondary causes of cholangitis. Since the aetiology of PSC remains poorly defined, medical therapy is currently limited to symptom improvement and prolonged survival. Ursodeoxycholic acid (UDCA), corticosteroids and immunosuppressants have been proposed alone or in combination to improve the clinical outcome. In selected cases, surgical or endoscopic procedures need to be considered. Orthotopic liver transplantation (OLT) is at the moment the only definitive approach although disease relapse has been reported. In this article the state of the art in PSC treatment and future promises in this field are reviewed.

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments.

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.

Computerisation of endoscopy reports using standard reports and text blocks.

BACKGROUND: The widespread use of gastrointestinal endoscopy for diagnosis and treatment requires effective, standardised report systems. This need is further increased by the limited storage of images, and by the need for structured databases for surveillance and epidemiology. We therefore aimed for a report system which would be quick, easy to learn, and suitable for use in busy daily practice. METHODS: Endobase III is an endoscopy information system offering three different ways of report writing, i.e. standard reports, text blocks and Minimal Standard Terminology (MST). A working group of two university and four general hospitals worked as a reference group for the development of standard reports and text blocks. Guidelines from various gastrointestinal endoscopy societies were followed to compose the reports. RESULTS: Standard reports were based on a list of distinct diagnoses; text blocks were based on anatomic landmarks and individual procedures. As such, 316 standard reports were developed for upper and lower gastrointestinal endoscopy, and endoscopic retrograde cholangiopancreatography (ERCP). In this way selecting one diagnosis produces a complete report. A total of 1571 different text blocks were additionally developed for each part of the gastrointestinal tract and for procedures during endoscopy. This module allowed generation of a full report on the combination of text blocks. Reports could be composed and printed within two minutes for 90% of cases. CONCLUSION: Standard reports and text blocks are a quick, user-friendly way of report writing accepted and used by a number of gastroenterologists in the Netherlands.

The effects of bile salt hydrophobicity on model bile vesicle morphology.

The addition of bile salts to vesicles supersaturated with cholesterol induces cholesterol precipitation, an important step in the formation of cholesterol gallstones. To investigate the effects of bile salt hydrophobicity on vesicle morphology, vesicles obtained from supersaturated model bile by density gradient ultracentrifugation, were incubated with mixtures of deoxycholate (DC) and ursodeoxycholate (UDC) with a constant total bile salt concentration of 30 mM but with a varying hydrophobicity index ranging from -0.31 (UDC alone) up to +0.72 (DC alone) depending on the composition of the mixture. Five days after addition of bile salts to vesicles, cholesterol precipitation was determined microscopically and incubation samples were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, mixed micelles, and cholesterol crystals. Structure and size of the isolated residual vesicles were studied by freeze fracture electron microscopy. The control, and samples exposed to hydrophilic bile salt mixtures, consisted of unilamellar vesicles of which more than 75% had a diameter of 50-80 nm. After addition of increasingly hydrophobic bile salt mixtures, multilamellar vesicles with progressively greater diameters (up to 1300 nm) were found, suggesting that vesicle fusion and aggregation took place and might hence be important in the cholesterol precipitation process. Accordingly, crystallization was positively correlated with bile salt hydrophobicity. We conclude that cholesterol crystallization from vesicles depends on the hydrophobicity of the bile salts added, and apparently occurs from fused or aggregated vesicles of extended magnitude and with a multilamellar constitution.

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.

Abnormalities of antroduodenal motility in type I diabetes.

OBJECTIVE: In the present study, a recently developed manometric technique was used to study antroduodenal motility in ambulant type I diabetic subjects. RESEARCH DESIGN AND METHODS: In 12 patients with type I diabetes, antroduodenal manometry was performed for 20 h during the fasting period and the postprandial period after a standardized dinner and breakfast. All patients had evidence of cardiac autonomic neuropathy and complained of dyspeptic symptoms. During the manometric study, the blood glucose levels were frequently monitored and kept close to euglycemia in the diabetic patients. The results were compared with 12 healthy control subjects. RESULTS: The migrating motor complex cycles observed in the diabetic subjects were longer than in the control subjects, 118.9 +/- 46.0 vs. 87.0 +/- 21.6 min (P < 0.05). This increase was attributable to a prolonged phase II, 78.0 +/- 35.5 vs. 37.7 +/- 18.5 min (P < 0.05). In the diabetic subjects, antral phase III was seen significantly less than in the control subjects, 16.7 vs. 43.3% (P < 0.005). In 50% of the diabetic patients, total absence of antral phase III was observed-this phenomenon was not seen in the healthy control subjects. After dinner, the antral motility index was less in diabetic subjects compared with the healthy volunteers, indicating antral hypomotility (P < 0.01). Six diabetic patients showed abnormal duodenal activity such as early recurrence of phase III and bursts after dinner. No significant differences in antral motility index or in duodenal motility patterns were observed after breakfast. Six diabetic patients complained of dyspeptic symptoms after dinner, whereas none had dyspeptic symptoms after breakfast. In 67% of the patients, nausea was reported after an early phase III or a burst. CONCLUSIONS: This study shows that prolonged ambulatory antroduodenal manometry is a feasible technique in patients. Recording multiple migrating motor complexes showed that interdigestive motor abnormalities of the stomach and duodenum are common in diabetic patients. Furthermore, it shows the occurrence of antral hypomotility and abnormal duodenal motility patterns after a high-calorie meal, with dyspeptic symptoms in diabetic patients being related to the composition of the meal.

Prevalence of delayed gastric emptying in diabetic patients and relationship to dyspeptic symptoms: a prospective study in unselected diabetic patients.

OBJECTIVE: Data on the prevalence of abnormal gastric emptying in diabetic patients are still lacking. The relation between gastric emptying and dyspeptic symptoms assessed during gastric emptying measurement has not yet been investigated. The aim was to investigate the prevalence of delayed gastric emptying in a large cohort of unselected diabetic patients and to investigate the relation between gastric emptying and gastrointestinal sensations experienced in the 2 weeks before and during the test meal, prospectively. RESEARCH DESIGN AND METHODS: Gastric emptying was evaluated in 186 patients (106 with type 1 diabetes, mean duration of diabetes 11.6 +/- 11.3 years) using 100 mg (13)C-enriched octanoic acid added to a solid meal. RESULTS: Gastric emptying was significantly slower in the diabetic subjects than in the healthy volunteers (T(50): 99.5 +/- 35.4 vs. 76.8 +/- 21.4 min, P < 0.003; Ret(120 min): 30.6 +/- 17.2 vs. 20.4 +/- 9.7%, P < 0.006). Delayed gastric emptying was observed in 51 (28%) diabetic subjects. The sensations experienced in the 2 weeks before the test were weakly correlated with the sensation scored during the gastric emptying test. Sensations assessed during the gastric emptying test did predict gastric emptying to some extent (r = 0.46, P < 0.0001), whereas sensations experienced in the previous 2 weeks did not. CONCLUSIONS: This prospective study shows that delayed gastric emptying can be observed in 28% of unselected patients with diabetes. Upper gastrointestinal sensations scored during the gastric emptying tests do predict the rate of gastric emptying to some extent and sensation experienced during daily life does not.

Effect of a standardized wheat bran preparation on serum lipids in young healthy males.

Feeding of a chemically standardized coarse wheat bran product in a dose of 0.5 g/kg body weight per day over a period of 4 weeks in young healthy male volunteers did cause a significant reduction in total serum cholesterol as well as in total serum triglycerides of 10 and 24%, respectively. Very low density lipoprotein-, high density lipoprotein-, and low density lipoprotein-cholesterol levels tended to diminish during bran feeding. The most marked reduction was observed in the high density lipoprotein- cholesterol fraction. Although the duration of this study was relatively short, it is concluded, that these results could have therapeutic consequences for the dietary management of hyperlipidemia. However, the lowering of high density lipoprotein-cholesterol could also be interpreted as unfavorable since an inverse relationship between high density lipoprotien-cholesterol levels and the occurrence of coronary heart disease has been established.

Effect of inhibition of gastric acid secretion on antropyloroduodenal motor activity and duodenal acid hypersensitivity in functional dyspepsia.

BACKGROUND: Heightened visceroperception and a decreased duodenal motor response to intraduodenal acid infusion have been reported in functional dyspepsia. AIM: To investigate the effect of treatment with a proton pump inhibitor on sensorimotor impairment in 19 patients with functional dyspepsia. METHODS: Patients were assigned double-blind to pantoprazole (n=10) or placebo (n=9) treatment for 2 weeks. Antropyloroduodenal manometry was performed before and after treatment, using a 21-channel catheter, and the responses to intraduodenal infusion of 5 mL of saline and acid were assessed. Nausea, fullness and epigastric pain were scored before and after each infusion. RESULTS: Acid induced a modest duodenal motor response and suppression of antral pressure waves, not altered by either treatment. However, acid evoked isolated pyloric pressure waves after pantoprazole treatment (P < 0.02), and not after placebo. Saline induced no motor response. Acid (not saline) induced nausea, both before and after treatment in both groups (all P < 0.05). Subgroup analysis of the seven acid-hypersensitive patients (37%) showed a tendency towards a decrease in nausea in all four pantoprazole-treated patients (P=0.07), in contrast to the three placebo-treated patients (P=1.0). CONCLUSIONS: In functional dyspepsia, pantoprazole influenced the acid-induced duodenogastric feedback mechanism, but not the impaired duodenal motor response. Duodenal acid hypersensitivity was decreased to some extent.

Effect of alosetron on left colonic motility in non-constipated patients with irritable bowel syndrome and healthy volunteers.

BACKGROUND: Alosetron is a 5-hydroxytryptamine-3 receptor antagonist reducing symptoms in female patients with diarrhoea-predominant irritable bowel syndrome, and is known to increase the colonic transit time. AIM: To study the effect of alosetron on left colonic phasic motility in ambulant non-constipated patients with irritable bowel syndrome and healthy volunteers. METHODS: In a double-blind, randomized, crossover design, 10 patients with irritable bowel syndrome and 12 sex- and age-matched volunteers were treated for two 7-day periods with alosetron, 4 mg b.d., or placebo b.d. On day 6 of each treatment period, a six-channel solid-state manometric catheter was positioned in the left colon and 24 h motility was studied on day 7. The periprandial phasic motility around dinnertime was evaluated in the descending and sigmoid colon. The high-amplitude propagated contraction frequency and characteristics were calculated. RESULTS: Alosetron appeared to increase the overall periprandial frequency in the sigmoid colon (P=0.043) and the mean amplitude of colonic contractions in the descending colon (P=0.007). The high-amplitude propagated contraction frequency was higher on alosetron during the second half of the day for patients with irritable bowel syndrome (P=0.002), with increased mean propagation length of high-amplitude propagated contractions (P=0.001). The stool frequency (P=0.024) and stool consistency score (P=0.002) were decreased by alosetron. CONCLUSIONS: The 5-hydroxytryptamine-3 receptor antagonist alosetron marginally increased left colonic periprandial phasic motility. Alosetron increased the number and propagation length of high-amplitude propagated contractions, which were paradoxically accompanied by a decrease in stool frequency and a firming of stool consistency.

Iron and inflammatory bowel disease.

Both anaemia of iron deficiency and anaemia of chronic disease are frequently encountered in inflammatory bowel disease. Anaemia of iron deficiency is mostly due to inadequate intake or loss of iron. Anaemia of chronic disease probably results from decreased erythropoiesis, secondary to increased levels of proinflammatory cytokines, reactive oxygen metabolites and nitric oxide. Assessment of the iron status in a condition associated with inflammation, such as inflammatory bowel disease, is difficult. The combination of serum transferrin receptor with ferritin concentrations, however, allows a reliable assessment of the iron deficit. The best treatment for anaemia of chronic disease is the cure of the underlying disease. Erythropoietin reportedly may increase haemoglobin levels in some of these patients. The anaemia of iron deficiency is usually treated with oral iron supplements. Iron supplementation may lead to an increased inflammatory activity through the generation of reactive oxygen species. To date, data from studies in animal models of inflammatory bowel disease support the theoretical disadvantage of iron supplementation in this respect. The results, however, cannot easily be extrapolated to the human situation, because the amount of supplemented iron in these experiments was much higher than the dose used in patients with iron deficiency.

The effect of omeprazole on gastro-oesophageal reflux and symptoms during strenuous exercise.

BACKGROUND: Strenuous exercise exacerbates gastro-oesophageal reflux and symptoms and this may be diminished by antisecretory medication with omeprazole. METHODS: Fourteen well-trained athletes (13 men, one woman), who indicated suffering from either heartburn, regurgitation or chest pain during competition running, performed two experimental trials at 2-week intervals using a randomized, double-blind, placebo-controlled crossover design. During the 6 days preceding the trial and on the trial day itself either 20 mg of omeprazole or a placebo was administered. Two hours after a low-fat breakfast and 1 h after the last study dose, the trial started with five successive 50-min periods: rest, three running periods on a treadmill, and recovery. Reflux (percentage time and number of periods oesophageal pH <4) was measured with an ambulant pH system during these periods. RESULTS: Compared to rest, reflux lasted significantly longer and occurred more frequently during the first running period, irrespective of the intervention, whereas during the second running period this effect was only observed with the placebo. Reflux occurred for longer and more frequently with the placebo than with omeprazole, but this was significant during the first two running periods only. Seven subjects reported heartburn, regurgitation and/or chest pain during exercise, irrespective of the intervention. Only a minority of the symptom periods was actually associated with acid reflux and in all cases this concerned periods with heartburn. CONCLUSIONS: Running-induced acid reflux, but not symptoms, were decreased by omeprazole, probably because most symptoms were not related to acid reflux.

A randomized trial in primary biliary cirrhosis comparing ursodeoxycholic acid in daily doses of either 10 mg/kg or 20 mg/kg. Dutch Multicentre PBC Study Group.

BACKGROUND: Ursodeoxycholic acid (UDCA) prolongs transplantation-free survival in primary biliary cirrhosis (PBC). However, the optimal therapeutic dose has not been established. AIM: To compare the effects of UDCA administered in daily doses of 10 vs. 20 mg/kg on symptoms, liver biochemistry and biliary UDCA enrichment. METHODS: A 6-month multicentre randomized open controlled trial was conducted to assess the effects of an increase in the dose of UDCA to 20 mg/kg/day vs. continuation of 10 mg/kg/day for patients who had not achieved biochemical normalization during treatment for at least 6 months with the 10 mg/kg dose. Clinical and laboratory evaluations were performed at entry and at 3-month intervals. The percentage UDCA in duodenal bile was assessed at entry and at 6 months. RESULTS: Sixty-one patients were enrolled. No side-effects of UDCA were observed. Within the 20 mg/kg/day group significant decreases were found for alkaline phosphatase (- 8%; P = 0.003), aspartate aminotransferase (- 11%; P = 0.01), alanine aminotransferase (- 17%; P < 0.001), gamma-glutamyl transferase (- 34%; P < 0.001), immunoglobulin M (- 11%; P = 0.002) and cholesterol (- 8.1%; P < 0.001). In the 10 mg/kg group none of these parameters differed significantly from baseline. No significant differences between dose groups for symptom scores or serum bilirubin were found. Biliary enrichment with UDCA increased from 37% to 46% in the 20 mg/kg group (P = 0.02) while remaining stable in the 10 mg/kg group. CONCLUSIONS: Liver biochemistry improved in PBC patients receiving UDCA 20 mg/kg/day compared to a dose of 10 mg/kg/day. Both doses were equally well tolerated. These results indicate that UDCA 10 mg/kg/ day is a suboptimal dose for treating PBC.

Increased prevalence of colonic adenomas in patients with acromegaly.

Forty-nine acromegalics and 57 controls matched for age and sex underwent colonoscopy. The control group consisted of patients investigated because of atypical abdominal complaints compatible with irritable bowel syndrome or constipation. The exclusion criteria for both groups included: age over 75 years, previous colonic polyps or cancer, previous colonic surgery, rectal blood loss, anemia, previous abdominal radiation, sigmoidoscopy, colonoscopy or barium enema performed for any indication within 3 years prior to the present study. Colonoscopy was successful in reaching the cecum in 72 and 77% of the controls and acromegalics, respectively (p = NS). Eleven (22%) of 49 acromegalics had biopsy-proven colonic adenomas versus only five (9%) of the control group (p < or = 0.05). Multiple adenomas were found in three of the 11 acromegalics and in none of the controls. In five of these 11 patients and in only one of the controls, at least one adenoma was located in the right colon. In addition, acromegalics tended to have larger adenomas. The group of acromegalics with and without adenomas did not differ significantly in age or duration of active disease. In conclusion, the present study shows that acromegalic patients have an increased risk of developing colonic adenomas.

Effects of ileal bile salts on fasting small intestinal and gallbladder motility.

In the fasting state, gallbladder emptying is related to phase III of the intestinal migrating motor complex. The effects of ileal infusion of mixed taurocholate-phospholipid micelles on fasting small intestinal motility (by a 17-channel catheter with side holes located in duodenum, jejunum and ileum) and gallbladder motility (by ultrasound) were investigated in eight healthy volunteers. After bile salt depletion by cholestyramine, 0.9% NaCl or mixed micelles were infused in the ileum during phase II of the migrating motor complex. Time to onset of subsequent phase III was significantly shorter after infusion of mixed micelles compared with 0.9% NaCl (32 +/- 5 min vs. 60 +/- 5 min, P = 0.01). Distal to the infusion port, numbers of pressure waves and their amplitudes were significantly lower during bile salt infusion compared with 15 min before infusion (11 +/- 6 per 15 min vs. 21 +/- 8 per 15 min, and 2.4 +/- 0.6 kPa vs. 2.8 +/- 0.5 kPa, respectively). Micellar infusions increased fasting gallbladder volumes to 170 +/- 5% of starting volumes (P < 0.0001). In conclusion, ileal infusion of mixed micelles influences the timing of phase III of the intestinal migrating motor complex, inhibits ileal motility and increases fasting gallbladder volumes. These findings may have important consequences for enterohepatic circulation of bile salts.

Acute intraduodenal bile salt depletion leads to strong gallbladder contraction, altered antroduodenal motility and high plasma motilin levels in humans.

Cholecystokinin is the main hormone involved in postprandial gallbladder contraction. There is also considerable gallbladder contraction in the fasting state, associated with phase III of the gastrointestinal migrating motor complex and release of the hormone motilin. It has been proposed that intraduodenal bile salts exert a negative-feedback control on postprandial cholecystokinin release and resulting gallbladder contraction. We wanted to elucidate whether a similar control mechanism on gallbladder contraction exists in the fasting state. We therefore performed gallbladder ultrasonography and 24-h antroduodenal motility registrations and determined plasma cholecystokinin and motilin levels in six healthy subjects before and after acute (4 g) and chronic (8 days; 8 g day(-1)) oral cholestyramine. Acute cholestyramine strongly decreased gallbladder volumes and increased motilin without changed cholecystokinin levels. There was a negative relationship between gallbladder volumes and plasma motilin levels. Although there was a persistent fasting pattern of antroduodenal motility, its cycle length was increased (P < 0.03) with markedly longer phase II (P < 0. 005). Fasting gallbladder volumes 24 h later were still strongly decreased but gradually increased to pretreatment levels. Before and after 8 days cholestyramine, interdigestive and postprandial gallbladder emptying, intestinal migrating motor complex and hormone levels did not differ. We conclude that acute (but not chronic) intraduodenal bile salt depletion with cholestyramine affects gallbladder and antroduodenal motility, possibly partly related to motilin release.

Differential effects of motilin on interdigestive motility of the human gastric antrum, pylorus, small intestine and gallbladder.

Motilin was infused in this study with the aim of examining refractory characteristics for motilin stimulation of antral phase III and fasting gallbladder emptying. Moreover, interdigestive pyloric and small intestinal motility from duodenum to ileum were studied, as these may be target organs for motilin. Eight fasting, healthy male volunteers received, on separate subsequent days, repeated infusions of 13leucine-motilin (8 pmol (kg min)(-1) for 5 min) or saline at 30 min after phase IIIs in the duodenum. Interdigestive motility of the antrum, pylorus, duodenum, jejunum and ileum was measured for maximum 10 h by using a 21-lumen perfused catheter. Gallbladder motility was measured by ultrasonography. Motilin infusions induced antral phase IIIs, but only after a preceding phase III of duodenal origin. Under this condition, time-interval to phase III at the duodenal recording site was 30 +/- 13 (SEM) min after motilin, compared with 79 +/- 14 min after saline (P < 0.01), and compared with 121 +/- 13 min for motilin infusion following an antral phase III (P < 0.001). Motilin did not affect small intestinal motility or isolated pyloric pressure waves (IPPWs). However, the number of IPPWs was significantly affected by the origin of the preceding phase III, irrespective of whether motilin or saline was infused. Gallbladder volume decreased significantly within 10 min after each motilin infusion. We conclude that this study clearly demonstrates differential regional effects of motilin. Motilin initiates antral phase IIIs, but stimulation is subject to a refractory period which is clearly prolonged after a preceding antral phase III. Motilin induced gallbladder emptying, however, is not subject to a refractory state. Small intestinal phase IIIs as well as pyloric IPPWs are not affected by motilin.

A simple reverse-phase high pressure liquid chromatographic determination of conjugated bile acids in serum and bile using a novel radial compression separation system.

A new HPLC method for analyzing conjugated bile acids in bile and especially in serum is presented. For separation of novel radial compression system with a 10-cm flexible-walled reverse phase column (RCM-100 Module, Waters Ass.) packed with micro-Bondapak C18 was used. Without any laborious hydrolysis and derivatization steps, 10 conjugated bile acids are separated easily with a mobile phase of methanol/0.01 mol/1 KH2PO4 (150:50, v/v; pH 6.0) in about 30 min, determined with a variable UV detector at 200 nm and a flow rate of only 0.5 ml/min. Dexamethasone was used as internal standard. Conjugated bile acids are extracted from serum with Sep-pak C18 cartridges after a 1:8 dilution with a mixture of the mobile phase and 0.2 mol/l NaOH (6:8, v/v). Extraction from bile was also performed using Sep-pak cartridges after a 1:20 dilution with 0.5 mol/l phosphate buffer (pH 7.0). The complete analysis time of the method, with Sep-pak extraction, is less than one hour. Recoveries for the different conjugated bile acid varied from 89.4% to 96.7% in serum. The HPLC method for serum and bile was validated for the primary bile acids cholate and chenodeoxycholate against the previously described GLC method. Agreement between the two methods was excellent. Duplicate analyses in serum and bile samples also showed that the reproducibility was good.

A bioluminescence assay for total 3 alpha-hydroxy bile acids in serum using immobilized enzymes.

A bioluminescence assay for bile acids was developed using a co-immobilized 3 alpha-hydroxysteroid dehydrogenase, diaphorase, and bacterial luciferase. The assay was specific for bile acids containing a free 3 alpha-hydroxyl group, as well as androsterone. Light output was linear over a bile acid concentration range of 1-20 000 pmol. Intra-assay precision was 6.2-8.2% and the recovery of added standards was 92-110%. Comparison of results using the bioluminescence assay with those using gas liquid chromatography revealed an excellent correlation (r = 0.99, n = 31). Since the bioluminescence assay is rapid, sensitive, specific, and uses inexpensive reagents, it appears to be an ideal method for the measurement of total bile acids in serum.

Bile acid and phospholipid fatty acid composition in bile of patients with cholesterol and pigment gallstones.

It has been previously reported that patients with cholesterol gallstones have increased biliary deoxycholate and arachidonate content as compared with normal subjects without gallstones. Increased biliary deoxycholate and arachidonate content might be a primary factor in the pathogenesis of cholesterol gallstones or merely an epiphenomenon due to the presence of gallstones. We therefore compared biliary bile acid composition in 46 patients with cholesterol gallstones and 22 patients with pigment stones. In addition, biliary phospholipid fatty acid composition was determined in 44 of these patients (30 cholesterol and 14 pigment stone patients). No significant differences were detected. In particular, the percentage deoxycholic acid (mean +/- SD: 20.3 +/- 8.8% and 21.5 +/- 10.9% respectively) and the percentage arachidonic acid (4.4 +/- 2.0% and 4.5 +/- 2.2%, respectively) were very similar. A significant correlation between age and biliary cholesterol saturation index was found only for the group of patients with pigment stones (R = 0.52, p less than 0.02). In conclusion, the present study does not support a primary role for increased biliary deoxycholic acid or arachidonic acid in the pathogenesis of cholesterol gallstones.