Vesicular cholesterol in bile. Relationship to protein concentration and nucleation time.

A study was done to determine whether the nucleation time was related to the amount of cholesterol carried in vesicles. Bile was obtained from cholesterol gallstone patients and controls. Gel-exclusion chromatography was used to separate vesicles and micelles in the native bile using an eluting buffer containing 10 mM sodium cholate. The percent of total cholesterol carried in vesicles in gallbladder bile of stone patients was significantly greater than that in control patients. Total cholesterol concentration in gallbladder bile of stone patients was significantly greater than in controls. This difference was due to the fact that vesicular cholesterol concentration was significantly greater in the gallbladder bile of stone patients compared to controls. Micellar cholesterol concentrations were similar in the two groups. Nucleation time was related significantly to vesicular cholesterol concentration in correlation analysis and, as previously shown, so was total protein concentration. This study supports the importance of vesicular cholesterol in solid crystal formation and demonstrates for the first time that the rate of cholesterol monohydrate crystal formation is directly related to the amount of cholesterol transported in vesicles.

Biliary micellar cholesterol nucleates via the vesicular pathway.

Biliary cholesterol nucleates primarily from phospholipid vesicles. In this study, we investigated the mode of nucleation of micellar cholesterol. Ten biles (four human and six model) were examined. The vesicular and micellar fractions of each bile were separated by gel chromatography. The whole biles and their isolated carriers were incubated at 37 degrees C until nucleation time. In whole human biles, the proportion of total cholesterol in vesicles rose throughout the incubation (from zero time to nucleation time) from 15.5 +/- 8.6% to 28.0 +/- 12.5%, and in model biles from 46.8 +/- 22.4% to 75.5 +/- 8.2%. The vesicular isolated fraction remained unchanged throughout incubation. In isolated micelles devoid of vesicles at zero time, new vesicles formed during incubation, carrying increasing proportions of cholesterol. At nucleation time, these vesicles contained 11.0% of originally micellar cholesterol in human biles, and 41.2% in model biles. The new vesicles formed in whole bile and in the micellar fraction were chromatographically and chemically similar to the vesicles originally present in bile. These data suggest that micellar cholesterol nucleates via the neoformation of phospholipid vesicles, which seem to be the final common pathway for cholesterol nucleation in bile.

Cholesterol-phospholipid vesicles in human bile: an ultrastructural study.

Phospholipid vesicles, a newly described (bile salt independent) mode of cholesterol transport in human bile, were previously characterized by quasi-elastic light scattering and gel filtration. In the present study the ultrastructure of these vesicles was investigated by electron microscopy using freeze-fracture and negative-staining techniques. Vesicles of varying size were found in all 14 hepatic and 3 gallbladder biles examined. The diameter of the vesicles ranged from 25 to 75 nm by electron microscopy after freeze fracture and from 54 to 94 nm by quasi-elastic light scattering. They had a spherical shape and appeared to be unilamellar. The appearance of the vesicles in fresh hepatic and gallbladder biles as well as in chromatographic fractions was similar. Vesicles were dissolved by the addition of exogenous bile salts. Cholesterol is transported in human bile by both vesicles and micelles. The role of the vesicles may be particularly important in preventing cholesterol precipitation in dilute and supersaturated biles.

Increased saturation of the fatty acids in the sn-2 position of phospholipids reduces cholesterol crystallization in model biles.

Changes in the molecular structure of biliary phospholipids were shown to have major effects on cholesterol solubility, carriers and crystallization in human and model biles. This study investigated systematically the effects of varying saturation of the phosphatidylcholine (PC) sn-2 fatty acid on the cholesterol crystallization process in 3 different model biles. Twenty % of the egg PC (EPC) in these biles were replaced by synthetic PC's with 16:0-18:0, 16:0-18:1, or 16:0-18:2 fatty acyl chains. With 18:0 in the sn-2 position, the crystal observation time (COT) was prolonged from 2 days in the control EPC solution to 14 days (p<0.05). The crystal growth rate (CGR) was reduced from 0.1 OD/day to unmeasurable levels, and the total crystal mass on day 14 decreased by 86%. The introduction of one (18:1), and two (18:2) double bonds in the sn-2 fatty acid rapidly reversed these effects. Ultracentrifugal analysis showed precipitable cholesterol as monohydrate crystals. In the 16:0-18:0 test solution, most of the precipitable cholesterol remained in the supersaturated multilamellar vesicles. Saturation of the biliary PC sn-2 fatty acyl chain prolongs the COT, slows the CGR, reduces the crystal mass, and extends cholesterol solubility in multilamellar vesicles. Desaturation of the sn-2 fatty acid reverses these effects.

Quantitation of phospholipid vesicles and their cholesterol content in human bile by quasi-elastic light scattering.

The proportion of biliary cholesterol carried by phospholipid vesicles may be an important determinant of the lithogenicity of bile. The distribution of biliary cholesterol between vesicles and other aggregational forms is often determined by gel filtration under standard conditions. The aim of this study was to measure the proportion of biliary cholesterol in vesicles in native unprocessed bile and to compare it with values obtained by chromatography. A modified quasi-elastic light-scattering method was used to measure vesicular cholesterol in whole bile. It was suitable only for lightly pigmented biles with a relatively monodisperse population of vesicles. In ten human biles examined, the proportion of cholesterol in vesicles by gel filtration was 40 +/- 8.1% (mean +/- S.D.) by chemical measurement, and 38 +/- 7.2% by [3H]cholesterol estimation. Quasi-elastic light-scattering measurements of these biles produced vesicular cholesterol values of 36 +/- 9.4%. Chromatography may affect lipid particles in bile. Nevertheless, it provides a relatively accurate measurement of biliary cholesterol in vesicles.

Phospholipid lamellae are cholesterol carriers in human bile.

Cholesterol solubility and precipitation in bile are major factors in the pathogenesis of cholesterol gallstones. At present, mixed micelles and phospholipid vesicles are considered to be the only cholesterol carriers in bile. In this study we present evidence showing that phospholipid lamellae are major cholesterol carriers in human bile. Lamellae are a known aggregational form in pure phospholipid model systems. In the present study, lamellae were demonstrated by electron microscopy after negative staining and by small-angle X-ray diffraction in all human gallbladder bile samples examined. During diffraction experiments, cholesterol was found to crystallize from these lamellae. Cholesterol carriers in bile were separated by high-resolution chromatography and by prolonged ultracentrifugation. Lamellae were shown to solubilize most of the biliary cholesterol; vesicles solubilized a lesser amount; while micelles solubilized only a minor portion. Our data suggest that phospholipid aggregates are the main cholesterol carriers in bile. Bile salts may control the equilibrium between the various aggregational forms of cholesterol-carrying phospholipids.

Nucleation of cholesterol from vesicles isolated from bile of patients with and without cholesterol gallstones.

Bile was obtained from patients with and without cholesterol gallstones at surgery. Biliary vesicles were separated from micelles by gel filtration. The cholesterol/phospholipid ratio in vesicles was much higher than in micelles. Cholesterol crystals nucleated from vesicular fractions, but nucleation from the micellar fractions was slow or did not occur at all. Cholesterol nucleated from vesicles obtained from bile of control patients as rapidly (2.4 days +/- 0.7) as from patients with stones (2.4 days +/- 0.9) and there was no difference in the vesicular cholesterol/phospholipid ratio. The effect of alteration of the bile salt environment was studied by changing the concentration of sodium cholate in the eluting buffer. At low concentrations (5 mM) only vesicles were eluted from the column. These vesicles had a relatively low cholesterol/phospholipid ratio and cholesterol nucleated slowly from these vesicles. At higher concentrations the proportion of micelles increased. The proportion of vesicles decreased progressively but their cholesterol/phospholipid ratio increased and the nucleation time fell. These studies demonstrate that cholesterol nucleates from vesicles in the absence of micelles, that control vesicles are not protected by tightly bound antinucleating substances and that exposure of vesicles to micelles strips relatively more phospholipid than cholesterol from the vesicular fraction, resulting in vesicles with higher cholesterol/phospholipid ratios and shorter nucleation times.

A non-micellar mode of cholesterol transport in human bile.

Quasi-elastic light scattering (QELS) was used to measure particle size in fresh human hepatic bile of 14 subjects. Particles with an approximate diameter of 700 A were found in all biles. The particles were almost unchanged after the bile salt concentration was reduced to 0.06 mM by dilution or dialysis against 150 mM NaCl. During dialysis bile salts were removed, while cholesterol and phospholipids remained in solution apparently in the large particles-vesicles. These experiments suggest the presence of a novel, bile salt-independent, mode of cholesterol transport in saturated human bile.

The induction of lamellar stacking by cholesterol in lecithin-bile salt model systems and human bile studied by synchrotron X-radiation.

Small angle X-ray scattering (SAXS) with synchroton radiation was used to investigate interactions among lipid particles in lecithin-bile salt model systems and in native gallbladder biles. In model systems in the absence of cholesterol, isotropic, continuous spectra were found, indicating the absence of periodic structures. In the presence of excess cholesterol, interaction in the form of lamellar stacking was detected by the appearance of discrete diffraction peaks. In the supersaturated cholesterol region of the commonly accepted phase diagram [1], where cholesterol crystals were expected, we found lamellar stacking. The high proportion of cholesterol to bile salts seems to be the common denominator of these models. The lamellar stacking was also found in native unprocessed bile. This effect of cholesterol on lipid structure has not been previously described. Lamellar stacking may contribute to cholesterol solubilization. Its influence on the kinetics of cholesterol crystallization is presently unknown.

Gallstones and diet in Tel Aviv and Gaza.

To study the possible association between diet and gallstones we investigated these two variables in two populations in Tel Aviv and Gaza with presumed differences in both. Gallstones were more frequent in Jews in Tel Aviv than in Arabs in Gaza, 12.1% versus 3.8% (p less than 0.001). This difference was fully accounted for by the 60+ age groups in both populations. There were no significant differences in the frequency of gallstones among the 20-39 and 40-59 age groups. Numerous and marked differences in diet composition were found between both populations. Energy, carbohydrate and fiber intake was higher in Gaza. The consumption of unsaturated fats was greater in Gazans and their P/S ratio was higher, 0.92 versus 0.70 in Tel Aviv. Beef and fish were usually eaten in Gaza while poultry was more frequently consumed in Tel Aviv. There were also differences in vitamin and mineral consumption. It cannot be determined which, if any, of these dietary differences is related to the lower frequency of gallstones in the older population of Gaza. Alternatively it could be the diet eaten in Gaza many decades ago, presumably poorer in protein and fat, which is responsible for these differences.

The absorption of low-dose methotrexate in patients with inflammatory bowel disease.

BACKGROUND: Recent clinical trials have demonstrated that methotrexate may have an important therapeutic role in the treatment of patients with inflammatory bowel disease, who are either refractory or intolerant to traditional medical therapy. The aim of this study was to evaluate the pharmacokinetics of low-dose oral methotrexate in patients with inflammatory bowel disease. METHODS: Methotrexate (12.5 mg) was given orally to nine patients with inflammatory bowel disease: five with Crohn's disease, and four with ulcerative colitis, and to six patients with rheumatoid arthritis who served as a control group. Blood samples were drawn at specific intervals to evaluate methotrexate plasma levels. RESULTS: Methotrexate was rapidly absorbed in all patients. Peak concentrations (Cmax) varied considerably, ranging from 0.25-0.87 micro M. The mean Cmax values were similar in all patient groups (0.59 +/- 0.12, 0.69 +/- 0.16 and 0.54 +/- 0.18 micro M, P not significant) for Crohn's disease, ulcerative colitis and rheumatoid arthritis, respectively. The mean area under curve in 120 min (AUC0-120) was also similar in all patient groups (32.9 + 11.3, 43.6 + 9.9 and 41.8 + 14.9 ng.min/mL, P not significant) for Crohn's disease, ulcerative colitis and rheumatoid arthritis, respectively. The mean time to reach Cmax, (tmax), varied between patient groups (84, 112 and 95 min, respectively, with a significant difference, P < 0.02, between the Crohn's disease and ulcerative colitis groups. A negative correlation was found between methotrexate dosage/kg and Cmax (r = -0.74) only in Crohn's disease patients but not in the other patient groups. CONCLUSIONS: Orally administered methotrexate is well absorbed in patients with inflammatory bowel disease including those with severe small bowel disease or resection. If methotrexate is proven to be effective in inflammatory bowel disease, it should be administered orally.

One week triple therapy with omeprazole, clarithromycin and tinidazole for Helicobacter pylori: differing efficacy in previously treated and untreated patients.

BACKGROUND: Triple therapy with omeprazole, clarithromycin, and tinidazole (OCT) has been found to be highly effective against Helicobacter pylori infection. However, its efficacy as a second line regimen for patients who failed metronidazole-based triple therapy has not been evaluated. AIM: The aim of this study was to evaluate the efficacy of low-dose, short-term OCT therapy in an Israeli population, and to compare results obtained in previously treated and untreated patients. METHODS: Patients with duodenal or gastric ulcers and chronic antral gastritis with H. pylori infection as assessed by rapid urease test and/or 14C urea breath test (14C-UBT), were studied. All patients received omeprazole 20 mg b.d., clarithromycin 250 mg b.d. and tinidazole 500 mg b.d. for 7 days. Eradication was assessed by 14C-UBT 4 weeks after treatment. RESULTS: One hundred and fourty-four patients (M/F = 81/63) were enrolled (mean age 48.1 years, range 12-78). Eradication of H. pylori was significantly different between patients who were initially treated with this regimen (90/94, 96%) and patients who had previously failed to eradicate H. pylori with standard triple therapy (27/50, 54%). Moreover, the eradication rate was significantly decreased in patients with more than one previous failure (9/22, 41%) compared to that in patients with only one failure (18/29, 62%). No other differences such as age, gastric pathology, ethnic origin, smoking habits, or pre-treatment urease activity were found to influence the eradication rate. CONCLUSIONS: One-week low-dose triple therapy with OCT is highly effective as an initial therapy in eradicating H. pylori infection. The efficacy is significantly lower when given as a second line treatment in patients who have previously failed to eradicate H. pylori with bismuth-based standard triple therapy.

Quantitative estimation of attenuation in ultrasound video images: correlation with histology in diffuse liver disease.

RATIONALE AND OBJECTIVES: To determine the relationship between the attenuation of backscatter intensity in B-scan images of the liver and diffuse liver disease in order to assess the usefulness of this method in providing quantitative objective characterization of diffuse liver diseases in general and in fatty liver in particular. METHODS: Twenty-four healthy volunteers and 28 patients with elevated liver enzyme levels who underwent liver biopsy were included in this study. An automatic far-field slope (FFS) algorithm that estimates the decrease in amplitude of the backscattered echo as a function of beam depth was implemented on the noncompensated image that was acquired on a commercial phased-array ultrasound system fitted to a custom-built interface card. The images were processed at a work-station. All scans were acquired repeatedly, read, and graded blindly by experienced ultrasound radiologists. Histology obtained via needle biopsy was reviewed without knowledge of the ultrasound findings. RESULTS: Analysis of the FFS data for fatty infiltration in all patient groups yielded a sensitivity of 67%, a specificity of 77%, a positive predictive value (PPV) of 77%, negative predictive value (NPV) of 67%, and an accuracy of 71%. The mean score of the ultrasound reviewers showed a sensitivity of 82%, a specificity of 66%, a PPV% of 68%, an NPV of 81%, and an accuracy of 72%. Normal FFS values (false-negative) were found in five patients with proved fatty infiltration. All of these patients had coexistent moderate to severe hepatic inflammation. However, FFS data in patients with uncomplicated (pure) fatty infiltration revealed a sensitivity of 100%, a specificity of 80%, a PPV of 89%, an NPV of 100%, and an accuracy of 92%. The best ultrasound score yielded a sensitivity of 100%, a specificity of 60%, a PPV of 80%, an NPV of 100%, and an accuracy of 85% in the same patients. CONCLUSIONS: The data demonstrate an excellent sensitivity (100%) of the FFS values in patients with uncomplicated fatty infiltration. This was also the only group of patients in whom the FFS score was superior to the radiologists' best score. The FFS method can be used as a tool to follow up the response to a clinical or research treatment and to obtain standardization of pattern interpretation independently of the individual reader.

A modified, solid phase radioimmunoassay for the differential diagnosis of acute and convalescent phases of hepatitis A infection.

The commercial assays for diagnosing the presence of hepatitis A antibodies (HAVAB; Abbott Laboratories, North Chicago, IL) or the presence of IgM class anti-hepatitis A virus antibodies (HAVAB-M; Abbott) do not provide precise information as to the timing of the acute infection. IgM class antibodies are detected as late as six months after the acute infection. In this study the authors describe a modified HAVAB test that inactivates the IgM class antibodies. It thus measures the proportion of IgG antibodies out of the total anti-hepatitis A virus antibodies. In a study of 139 patients with impaired liver function, the available and modified tests showed good agreement except for the convalescent phase of hepatitis A. During serial testing for three months after the acute infection, the commercial tests continuously detected IgM class antibodies. The modified test detected predominantly IgG class antibodies from four weeks on. By six weeks, 85% of the patients had predominantly IgG class antibodies. The modified test thus provides information on the timing of recent hepatitis A infection.

Morphological transformation of Helicobacter pylori during prolonged incubation: association with decreased acid resistance.

The survival of clinical isolates of H pylori at two cultural ages (two and four days) at pH 2, in the presence of different buffers, with and without urea, was investigated. It was found that the morphological changes which occur with longer incubation of H pylori have an inverse correlation with its resistance to an acidic environment. The finding that the addition of urea almost reversed this phenomenon and prolonged survival of the cultures emphasises the role of urea in the survival of H pylori in acidic environments.

Imaging supramolecular aggregates in bile models and human bile.

Investigation of cholesterol crystallization is essential for the understanding of gallstone formation. Previous work has revealed a variety of aggregates of different sizes and shapes prior to the appearance of "classical" plate-like cholesterol monohydrate crystals both in native biles and model systems. In this article, we review existing data based on various microscopic techniques and present data on microstructural pathways leading to cholesterol crystal formation in two different bile models and in native bile. In continuation of our recent investigation of microstructures in nucleating human bile, we now present data suggesting that polymorphism is not limited to complex native bile, but also appears in two, simplified model systems. These studies employed cryo-transmission electron microscopy (cryo-TEM) and video-enhanced light microscopy, using Nomarski optics (VELM). Only the combined use of these two complementary, non-perturbing direct methods can cover the whole range of microstructures ranging from a few nanometers to several microns. Concentrated isotropic solutions of bile models, composed of cholesterol, lecithin and taurocholate, were diluted to induce cholesterol supersaturation and start an evolution of microstructures, leading to cholesterol crystallization. Initially, small spheroidal micelles were observed by cryo-TEM. Subsequently, uni-, oligo- and multilamellar vesicles, compatible with structures seen at the same time by VELM, appeared in coexistence with micelles. Thereafter, during a dynamic phase of cholesterol crystallization, filaments, tubular and helical microstructures, as well as classical plate-like cholesterol monohydrate crystals were noted by light microscopy. Eventually, large plate-like crystals were observed by VELM, while cryo-TEM revealed only small spheroidal micelles. The crystallization process in native human bile during ex vivo incubation was found to bear close resemblance to the findings in the model systems, further supporting the applicability of these systems to the exploration of microstructural aspects of nucleating human bile.

Prostaglandin E2 in pyloric stenosis.

Prostaglandins are presumed to have many cytoprotective properties that play a role in the pathogenesis of duodenal ulcer and its complications where decreased levels of prostaglandin E2 (PGE2) impair gastric motility, oppose ionic membrane influx, and enhance obstructive changes. These are just some of the mechanisms that may cause pyloric obstruction and may result from decreased PGE2 levels. To evaluate this hypothesis, 17 patients with duodenal ulcer complicated by pyloric stenosis were examined. Biopsy specimens were obtained from the duodenal bulb, ulcer margins, gastric antrum, fundus, and gastric secretions. Prostaglandin E2 levels were measured and compared with those taken from the same areas during a second endoscopy in a later quiescent or exacerbated phase. During the active phase of pyloric stenosis, decreased levels of PGE2 were found in the gastroduodenal tissues and secretions were compared with levels found during convalescence. These level differences were statistically significant. A correlation between the severity of the clinical and endoscopic findings and the PGE2 levels was found. A further decrease in PGE2 levels in the second endoscopy were indicative of the presence of scar tissue, representing an irreversible obstructive peptic disease.

Lithogenicity of human bile is reduced by freezing and thawing.

We examined the effects of freezing and thawing upon the nucleation time and the distribution of cholesterol between micelles and vesicles in 9 human gallbladder and 7 hepatic biles. The nucleation time was significantly longer after freezing when compared to fresh samples (22.4 +/- 2.6 vs. 7.4 +/- 1.9 days, respectively). Concomitantly, a substantial shift of cholesterol from vesicles to micelles was noted, with the proportion of vesicular cholesterol decreasing from 26.5% +/- 6.0% in fresh biles to 8.6% +/- 2.3% after freezing. These effects were observed in all types of human biles, regardless of origin, cholesterol saturation or initial presence of cholesterol crystals, and were most notable after the first week of freezing. The decrease in vesicular cholesterol in all biles and the increase in nucleation time of gallbladder biles correlated with the time the samples had been in a frozen state. It is concluded that the lithogenic properties of human bile are not maintained during storage at -20 degrees C. Freezing results in a shift of cholesterol from vesicles to micelles and reduces the tendency of cholesterol to crystallize from bile samples.

Passive smoking in patients with inflammatory bowel disease: an Israeli multicentre case-control study.

BACKGROUND: The association between smoking and inflammatory bowel disease (IBD) is well established. There are, however, no large scale studies of passive smoking in inflammatory bowel disease and this has never been surveyed in the Jewish population of Israel. AIM: To study the passive smoking exposure of Jewish IBD patients in Israel in a large scale multicentre study. METHODS: Patients with established IBD, aged 18-70 years, were interviewed regarding smoking and other habits. Two controls, one clinic and one neighbourhood, matched by age, sex, community group, and education, were sought for each subject. RESULTS: Five hundred and thirty-four patients (273 ulcerative colitis (UC) and 261 Crohn's disease (CD)), 478 clinic controls and 430 community controls were interviewed. There were no significant differences in the passive smoking habits between IBD patients and their controls. Fifty-one percent of UC patients, 50% of the clinic controls and 58% of the community controls were exposed to passive smoking at home (NS); similar results were found among CD patients (50%, 55% and 56%, respectively). When a quantitative exposure index was used UC patients were significantly less exposed to passive smoking than were their community controls (7.46 +/- 8.40 vs 9.36 +/- 9.46, n = 229, P< 0.031). There was no difference in the exposure to passive smoking among CD patients and their controls. No differences in exposure to passive smoking were found when UC patients who had never smoked were compared with their controls. When the quantitative index was used 'never-smoked' CD patients tended to be less exposed to passive smoking at home than their community controls (5.40 +/- 7.60 vs 8.04 +/- 8.72, P < 0.05). CONCLUSION: There is a lack of association between passive smoking and IBD in Jewish patients in Israel. When a quantitative exposure index was used UC patients were found to be less exposed to passive smoking than their community controls.