Cholesterol Monohydrate Crystals Research Articles

Occult microlithiasis in ‘idiopathic’ acute pancreatitis: Prevention of relapses by cholecystectomy or ursodeoxycholic acid therapy

Gallstone pancreatitis is usually related to small stones, which may not be detected by conventional cholecystographic techniques. In the current study, it was hypothesized that some patients with acute pancreatitis of unknown cause could harbor occult microstones in the gallbladder. Therefore, evidence was sought prospectively of missed gallstones by biliary drainage and microscopic examination of centrifuged duodenal bile in 51 patients recovering from an attack of acute pancreatitis, including 24 patients with relapsing episodes. Clusters of cholesterol monohydrate crystals, calcium bilirubinate granules, and/or CaCo3 microspheroliths were found in 67% of the patients. Biliary drainage showed no abnormal findings in 12 patients convalescing from a bout of known alcoholic pancreatitis. Examination of gallbladder bile at cholecystectomy and/or serial ultrasonography of the gallbladder for up to 12 months showed that 73% of the patients with unexplained pancreatitis had biliary sludge or microlithiasis; the prior finding of biliary crystal/solid markers predicted their existence with both a sensitivity and a specificity of 86% and a predictive value of 94%. The probability of harboring occult gallstones was also associated with age (P = 0.004), prior recurrent pancreatitis (P = 0.024), and altered liver function tests results during an index episode (P = 0.003). In 13 patients with cholesterol monohydrate crystals in bile, ursodeoxycholic acid (10 mg · kg−1 · day−1) eliminated gallbladder microlithiasis within 3–6 months, and subsequent maintenance treatment with a daily dose of 300 mg prevented both gallstone recurrence and further attacks of pancreatitis over a mean follow-up period of 44 months. Cholecystectomy also prevented gallstoneassociated relapses in 17 of 18 patients followed up for a mean postoperative period of 36 months. This study provides firm evidence showing that in most patients with idiopathic acute pancreatitis, the disease is related to microscopic gallstones, as evidenced by the follow-up development of macroscopic stones or sludge and by the prevention of relapses with either cholecystectomy or a cholelitholytic bile acid. Occult gallstones should be strongly suspected when acute pancreatitis of unknown cause occurs in a relapsing manner and in aged patients and when it is associated with altered liver function test results. Biliary microscopy and/or follow-up ultrasonography of the gallbladder provide a simple means of uncovering them to institute appropriate therapy and prevent further attacks.

Gallstones in cystic fibrosis: A critical reappraisal

Radiolucent gallstones are common in young adults with cystic fibrosis. In the mid-1970s, it was suggested that gallstones are made of cholesterol, but this hypothesis has never been tested. Several recent studies have shown that the detection of cholesterol monohydrate crystals in bile has high sensitivity and specificity for the diagnosis of cholesterol gallstones. We therefore used this approach to study 17 young adults with cystic fibrosis, 10 of whom had radiolucent gallstones. The two groups of patients were comparable in age and gender (all patients but one were male). Duodenal bile was obtained after gallbladder contraction with intravenous cerulein; it was used for lipid and protein chemistry studies and for polarizing microscopy. The latter was performed both in whole bile and in the postultracentrifugation (100,000 g) sediment. Bile cholesterol saturation did not significantly differ between patients with (1.21 +/- 0.28) or without gallstones (0.99 +/- 0.54). Slight cholesterol supersaturation was found in 7 of 10 gallstone and three of seven nongallstone patients. At no time were cholesterol crystals detected in either the group, even after bile ultracentrifugation. Two more cystic fibrosis patients with gallstones died of severe bronchopneumopathy, and small pigment gallstones were obtained at autopsy. At stone analysis, cholesterol content was 44% and 28% of dry weight, respectively. Infrared spectroscopy of stone powder was compatible with the presence of calcium bilirubinate and proteins as major components. We conclude that radiolucent gallstones of cystic fibrosis are not of the conventional cholesterol type.

Inhibition of prostaglandin synthesis fails to prevent gallbladder mucin hypersecretion in the cholesterol-fed prairie dog

Gallstone formation in the cholesterol-fed prairie dog is preceded by an increase in mucin secretion by the gallbladder epithelium, and mucin hypersecretion is believed to promote cholesterol gallstone formation by accelerating the nucleation of cholesterol monohydrate crystals. Some studies have suggested that gallbladder mucin hypersecretion is mediated by increases in gallbladder prostaglandin synthesis, but other observations are difficult to reconcile with this view. An organ culture technique was used to measure mucin secretion in normal prairie dog gallbladder in response to exogenous prostaglandins and agents that increased or decreased endogenous prostaglandin production. Incubation with indomethacin produced a concentration-dependent inhibition of endogenous prostaglandin synthesis with virtually complete inhibition at 10−5 mol/L indomethacin. However, indomethacin had no effect on gallbladder mucin secretion at concentrations as high as 10−5 mol/L, and significant inhibition of mucin secretion was only found at 10−4 mol/L indomethacin, a concentration that also produced a significant increase in lactate dehydrogenase release from cultured explants. Incubation of gallbladder explants with the calcium ionophore A23187 significantly stimulated endogenous prostaglandin synthesis in a concentration-dependent manner, increasing synthesis of prostaglandins E and F to as much as 278% ± 20% and 335% ± 21% of basal values, respectively; however, the same concentrations of A23187 did not stimulate mucin secretion. Incubation of gallbladder explants in the presence of exogenous prostaglandin E2 or prostaglandin F2a in concentrations as high as 10−6 mol/L also did not stimulate mucin secretion. Prairie dogs fed a lithogenic 1.2% cholesterol diet showed a significant increase in gallbladder mucin secretion after 1 week (117.5 ± 10.2% of control, P < 0.05), and 4 of 5 had formed cholesterol monohydrate crystals after 3 weeks. Long-term treatment with indomethacin, 1.2 mg · kg−1 · day−1, failed to inhibit gallbladder mucin hypersecretion (129.2 ± 10.7% of control after 1 week) or cholesterol monohydrate crystal formation (3/5) in cholesterol-fed prairie dogs. Furthermore, incubation of explants with 10−5 mol/L indomethacin failed to prevent in vitro mucin hypersecretion in cholesterol-fed animals. These findings suggest that prostaglandins do not regulate gallbladder mucin secretion in the prairie dog, and it is unlikely that increases in gallbladder prostaglandin synthesis are responsible for mediating gallbladder mucin hypersecretion during cholelithiasis in the prairie dog.

Ceftriaxone-associated gallbladder sludge: Identification of calcium-ceftriaxone salt as a major component of gallbladder precipitate

Ceftriaxone, a third-generation cephalosporin, is partially excreted into bile. With its clinical use, the formation of gallbladder sludge detected by ultrasonography has been reported. Four surgical specimens were examined and no gallstones were found. Instead, fine precipitates of 20–250 μm were present. Microscopically, there was a small number of cholesterol monohydrate crystals and bilirubin granules among an abundant amount of granular-crystalline material that was not morphologically cholesterol monohydrate crystals. The chemical composition of the precipitates (n = 4) was determined. There was a small amount of cholesterol (1.7% ± 0.8%) and bilirubin (13.9% ± 0.74%). The major component of the precipitate was a residue. On further analysis using thin-layer chromatography, high-performance liquid chromatography, and electron microprobe analysis, the residue was identified as a calcium salt of ceftriaxone. The residue also had identical crystal morphology and chromatographic elution profile as authentic calcium-ceftriaxone standards. It is concluded that ceftriaxone, after excretion and being concentrated in the gallbladder bile, can form a precipitate. The major constituent has been identified as a ceftriaxone-calcium salt.

Bilirubin monoglucuronide promotes cholesterol gallstone formation

Recent evidence suggests that cholesterol (Ch) solubility in bile is determined by a complex interaction of mixed micelles and lecithin-cholesterol vesicles. Bilirubin monoglucuronide (BMG), which binds to bile salts and incorporates into mixed micelles, may displace cholesterol from micelles into vesicles, thus favoring cholesterol monohydrate crystal precipitation. Therefore, we designed an experiment to test the hypothesis that BMG may enhance cholesterol gallstone formation without inducing cholesterol supersaturation. For 8 weeks, 28 adult male prairie dogs were fed either a control, nonlithogenic diet (0.03% Ch), a high carbohydrate diet (CHO) which has no cholesterol but increases hepatic bilirubin secretion, or the same CHO diet plus 0.03% Ch. Cholecystectomy was then performed, and bile was examined microscopically for stones or crystals and analyzed for BMG and biliary lipids. Cholesterol saturation index was calculated. Cholesterol gallstones were found in none of the control animals and in 13% of the CHO-fed animals. However, the addition of trace cholesterol to the CHO diet resulted in an 88% incidence of cholesterol gallstones ( P < 0.001 vs control, P < 0.01 vs CHO, respectively). Gallbladder bile was unsaturated with cholesterol in all groups. (control = 0.65 ± 0.05, CHO = 0.46 ± 0.05, CHO + 0.03% Ch = 0.70 ± 0.03). CHO feeding alone or with trace cholesterol significantly elevated gallbladder bilirubin monoglucuronide, phospholipid, and cholesterol concentrations when compared to controls. These data suggest that in the prairie dog a high carbohydrate diet with only trace amounts of cholesterol increases bilirubin monoglucuronide in gallbladder bile and causes cholesterol gallstone formation without inducing cholesterol supersaturation. We conclude that bilirubin monoglucuronide may promote cholesterol precipitation by displacing cholesterol from mixed micelles into lecithin-cholesterol vesicles.

Different hepatobiliary effects of oral and transdermal estradiol in postmenopausal women

Estrogen-replacement therapy is important for the prevention of postmenopausal osteoporosis. However, oral synthetic and conjugated estrogens increase biliary cholesterol saturation index and risk of gallstone disease. To examine whether transdermal estrogen administration could avoid these adverse effects, 17 postmenopausal women were treated with transdermal estradiol (Estraderm TTS; Ciba-Geigy, Arnhem, The Netherlands), 100 μg/day for 4 weeks, and after 1 month without therapy, with oral estradiol (Progynova; Schering, Weesp, The Netherlands), 2 mg/day for 4 weeks. The increase in the serum estradiol level was much higher during transdermal than oral estradiol administration. On the contrary, the increase in the serum estrone level was much more pronounced during oral treatment. Both modes of treatment led to a similar reduction of urinary calcium excretion. A highly significant decrease in serum phosphate levels was found during transdermal therapy. Biliary cholesterol saturation index did not change during transdermal therapy (mean ± SEM, 1.25 ± 0.06 before and 1.22 ± 0.07 at the end of transdermal therapy; P = NS). A slight increase in cholesterol saturation index that did not reach statistical significance was found during oral therapy (1.28 ± 0.09 before and 1.36 ± 0.09 during oral treatment). However, the subgroup of women with strong increases in serum estrone levels during oral estradiol therapy (>0.5 pmol/mL; n = 8) generally had increased biliary cholesterol saturation index, a decrease in relative percentage chenodeoxycholic acid in bile, and increased serum sex hormone-binding globulin levels during oral treatment. Cholesterol monohydrate crystals were never found in duodenal biles during either treatment. This study indicates that transdermal estradiol does not induce lithogenic bile. On the contrary, oral estradiol leads to lithogenic bile in a subgroup of women with strong increases in serum estrone levels during oral treatment.

Comparison of gall bladder bile and endoscopically obtained duodenal bile.

In 10 patients with gall stone disease (eight women, two men; mean (SD) age 47.4 (13) years), bile was obtained by endoscopic aspiration after stimulation of the gall bladder with ceruletid and also by fine needle puncture of the gall bladder under local anaesthetic. The total lipid concentration of the puncture bile samples was mean (SD) 11.9 (4.7) g/dl, significantly higher than the endoscopic bile samples (3.9 (3.3) g/dl, p less than 0.001). Total bile acids, phospholipids, and biliary cholesterol (expressed in mol%) and cholesterol saturation index showed no significant differences between the two types of samples. The glycocholic acid concentration in the endoscopically obtained bile (27.7 (6.6) mol% v 23.3 (5.4) mol%; p less than 0.01) was significantly higher than the puncture bile samples. Puncture bile exhibited a significantly shorter nucleation time (3.5 (3.3) days v 19.6 (11.9) days; p less than 0.001). For determination of the nucleation time, endoscopic bile aspiration after gall bladder stimulation with ceruletid led to adequately concentrated samples in 50% of the study subjects. Cholesterol monohydrate crystal formation in native bile was observed in six samples of puncture bile and in three samples of the endoscopically obtained bile. The presence of cholesterol crystals and the determination of nucleation time in the puncture bile were the best discriminants between cholesterol and pigment gall stones and correlated well with computed tomogram analysis.

Biliary Sludge

Biliary sludge has been for many years a poorly defined entity, usually with low amplitude, nonshadowing echoes within the most dependent part of the gallbladder, which shift under the influence of postural changes. From a sonographic point of view, the detection of sludge implies the coexistence of small-sized, solid components and of a gel-like embedding material. The chemical nature of biliary sludge has recently been recognized to be predominantly composed of a coaggregate of cholesterol monohydrate crystals and liquid crystalline droplets, and in some cases, such as obstructive jaundice or symptomatic liver diseases, by bilirubin granules, all embedded in a gel matrix of mucous glycoproteins. From a pathogenic point of view, biliary sludge is often associated with biliary stasis, or with conditions characterized by impaired gallbladder contraction, such as prolonged total parenteral nutrition, fasting, and pregnancy. Other causes include mucus hypersecretion, which may favor cholesterol nucleation and crystal growth, and bile infection. Sludge may be an intermediate step in the formation of different types of stones. From an epidemiological point of view, sludge is quite rare in the asymptomatic, free-living population, but may be common in selected series of symptomatic patients. From a clinical point of view, sludge often has a fluctuating course, including frequent disappearances and reappearances, suggesting that the early stages of gallstone formation are reversible.

Effect of dietary fish oil on coronary artery and aortic atherosclerosis in African green monkeys.

Studies were carried out for 2.5 to 3 years in adult male African green monkeys (grivet subspecies) fed diets containing 22% of calories as lard or fish oil with 40% of calories as fat and 0.75 mg cholesterol/Kcal to determine if isocaloric substitution of menhaden fish oil for lard affects coronary artery atherosclerosis. The average total plasma cholesterol concentrations during the experimental period were significantly lower for the fish-oil group (231 +/- 37 mg/dl) compared to the lard group (360 +/- 44 mg/dl), but this difference did not become apparent until after 5 months of experimental diet consumption. High density lipoprotein cholesterol concentrations were 30% lower (p less than 0.01) for the fish-oil group also (57 +/- 5 vs. 82 +/- 6 mg/dl). Plasma triglyceride concentrations were low for both groups, but after about 5 months of diet consumption, they were higher for the animals fed fish oil (25 +/- 2 mg/dl) compared to their lard-fed counterparts (15 +/- 1 mg/dl). Coronary artery intimal area (in this case a measure of early atherosclerotic lesion size) was low in all animals but was significantly less (p less than 0.03) for the fish oil vs. lard groups (0.01 +/- 0.002 vs. 0.03 +/- 0.009 mm2). More atherosclerosis was found in other arteries, and a trend was seen of less atherosclerosis in the thoracic aorta and common carotid arteries of the fish-oil group. The size of lesions in the abdominal aorta was similar between diet groups, but microscopic examination of arteries of the lard group revealed relatively more cholesterol monohydrate crystals compared to the arteries of the fish-oil group. Chemical analysis showed that there was less esterified cholesterol (1.46 +/- 0.71 vs. 3.43 +/- 0.74 mg/g, p = 0.04) and free cholesterol (3.7 +/- 2.15 vs. 7.05 +/- 1.68 mg/g, p = 0.08) in the abdominal aortas taken from the animals fed fish oil. There was a significant correlation between low density lipoprotein (LDL) cholesteryl ester (CE) fatty acid ratio (i.e., saturated + monounsaturated/polyunsaturated species) and the amount of esterified (r = 0.59) and free (r = 0.63) cholesterol in the abdominal aortas. Compared to the lard group, animals fed fish oil had significantly lower LDL CE melting temperatures (26 +/- 1 vs. 38 +/- 1 degree C) and significantly smaller LDL particles (2.68 +/- 0.10 vs. 3.25 +/- 0.38 g/mumol). Therefore, the potentially antiatherogenic effects of dietary fish oil include its ability to decrease the concentration, size, CE content, and CE melting temperature of plasma LDL.

Altered bile composition during cholesterol gallstone formation: Cause or effect?

Gallbladder stasis, increased gallbladder absorption, and elevated biliary levels of calcium, hydrogen ion, and bilirubin have been implicated as factors potentially critical to cholesterol crystal precipitation. Previous studies, however, have analyzed bile only when crystals or gallstones have already formed. Therefore, we tested the hypothesis that changes in bile composition are a late effect, occurring only after crystal formation. Adult male prairie dogs were fed a standard nonlithogenic control diet ( n = 7) or a lithogenic 1.2% cholesterol diet for 5, 9, or 14 days to cause cholesterol saturation ( n = 7), cholesterol monohydrate crystals ( n = 7), or gallstones ( n = 7). Gallbladder bile was examined microscopically for crystals, and analyzed for ionized calcium, bilirubin, pH, total protein, and biliary lipids. The ratio of gallbladder to hepatic bile radiolabeled cholic acid specific activity ( R sa) was calculated as an index of gallbladder stasis. Cholesterol saturation index was calculated. The results demonstrate that increased gallbladder bile cholesterol saturation and total protein concentration precede cholesterol monohydrate crystal precipitation. However, changes in gallbladder ionized calcium, unconjugated bilirubin, pH, stasis, and absorption were noted only after crystals and gallstones had already formed. These data indicate that alterations in gallbladder bile calcium, bilirubin, pH, stasis, and absorption are not early changes, but occur simultaneously with or after crystal formation. Increased biliary protein, however, which was elevated prior to nucleation, may be an important mediator of cholesterol precipitation in cholesterol-saturated bile.

Experimental ultrasonic angioplasty: Disruption of atherosclerotic plaques and thrombi in vitro and arterial recanalization in vivo

To investigate the use of high energy ultrasound as an alternative energy for angioplasty, an experimental ultrasonic angioplasty device was developed. The device was studied in two bioassay systems: an in vitro system for the disruption of atherosclerotic plaques and thrombi and an in vivo system for the recanalization of occluded canine femoral arteries.In vitro, sonication efficiently reduced the size of the plaques. Atheromatous plaques (n = 11) disrupted at a rate of 21 ± 8 s/cm2; complicated plaques (n = 14) disrupted at a rate of 132 ± 45 s/CM2 (p < 0.001). Histologic examination revealed that the disruption of the plaques took place without concurrent damage to the media or adventitia. Ninety percent of the disrupted plaque debris had a diameter of <20 μm and was composed primarily of cholesterol monohydrate crystals. Solid thrombus (n = 5) weight was reduced from 1.6 ±0.2 to 0.4 ± 0.1 (p<0.0001) after 20 s of sonication.In vivo, sonication resulted in recanalization in all seven arteries tested in seven dogs. The obstruction was reduced from 93 ± 11% to 18 ± 7% (p < 0.001). On histologic examination, the arterial wall injury index was found to be 1.56 ± 0.42 in the test arteries compared with 1.37 ± 0.47 in the control arteries (p = NS).The disruption of atherosclerotic plaques and thrombi, together with the efficient recanalization of the occluded arteries, demonstrates the potential of ultrasound angioplasty as a catheter-based technique for angioplasty. The histologic findings suggest that the ultrasonic angioplasty device selectively disrupts the ultrasound-sensitive atherosclerotic plaques and thrombi, with minimal damage to the ultrasound-resistant arterial wall.

High-resolution electron microscopic investigation of crystalline cholesterol monohydrate in human atheroma at the atomic level

High-resolution electron microscopic investigation of cholesterol monohydrate crystals obtained from human atheroma was carried out for the purpose of characterization of the crystal lattice, demonstration of crystallization processes and identification of crystal disorders. By high-resolution electron microscopy the crystal structures of perfect cholesterol monohydrate crystals were characterized as regular lattice arrays which consisted of stacks of repetitive rod-shaped substructures ca 1.58 nm long and 0.16 nm wide, with the total thickness of bilayered substructures ca 3.36 nm. These substructures were in an end-to-end arrangement of approximately side-to-side parallel packing, with a centre-to-centre spacing ca 0.32 nm. At the atomic level the lattice arrays were made up of regularly spaced rows of dots ca 0.28 nm × 0.16 nm in size. These dots possessed a six-fold ring-like shape, and were arranged in a hexagonal structure with an additional dot in the centre. High-resolution electron microscopic observations of the partially crystallized particles of cholesterol monohydrate showed various stages of cholesterol crystallization, from very small short-ordered segment of lattice arrays to different sized nano- and microcrystallites in the amorphous matrix of the crystals. Furthermore, crystal growth was also demonstrated from detailed examination of the crystal surfaces, the interfaces between the crystals and the boundary structures between the amorphous and crystalline phases. In addition, high-resolution electron microscopy could clearly identify various kinds of crystal defect in the cholesterol monohydrate crystals, including considerable variations of lattice spacings with focal fragmentation of lattice fringes, derangement of atom-sized dots along the lattice fringes and marked alterations of the morphology of atom-sized dots with the vacancies along the lattice arrays. It is hoped that such information obtained from high-resolution electron microscopic observations of the crystalline cholesterol in human atheroma at the atomic or near-atomic level may be helpful by providing a more complete understanding of the pathogenetic mechanisms responsible for the formation, progression and regression of the acellular lipid-rich cores of advanced atherosclerotic plaques.

Bile Concentration Is A Key Factor for Nucleation of Cholesterol Crystals and Cholesterol Saturation Index in Gallbladder Bile of Gallstone Patients

We investigated whether bile concentration influenced cholesterol saturation index or nucleation time of cholesterol monohydrate crystals in a large number of gallbladder bile samples. Pigment stone patients never had cholesterol crystals in their fresh biles, and nucleation time was always longer than 20 days. Of the cholesterol stone patients 79% had cholesterol crystals in their fresh biles. Long nucleation times were generally found in cholesterol stone patients with dilute biles despite a high cholesterol saturation index. Nucleation time was usually short if bile was well concentrated despite a relatively low saturation index. Serial in vitro dilution of concentrated biles from cholesterol gallstone patients resulted in progressively prolonged nucleation times. Patients with solitary cholesterol stones had longer nucleation times than patients with multiple cholesterol stones. This study indicates that bile concentration is an important factor for nucleation time and cholesterol saturation index. Moreover, solitary and multiple cholesterol stones may have a different pathogenesis.

Effects of dietary fish oil on biliary phospholipids and prostaglandin synthesis in the cholesterol‐fed pairie dog

Cholesterol gallstone formation in the prairie dog is accompanied by an increase in the percentage of biliary phospholipids containing arachidonic acid, and an increase in gallbladder prostaglandin (PG) synthesis, but the pathogenetic significance of these changes is unclear. Dietary supplementation with eicosapentaenoic acid (EPA), an omega-3 fatty acid which is commonly found in fish oil, decreases prostaglandin synthesis in some tissues by replacing arachidonic acid, and by competitively inhibiting prostaglandin synthesis. We studied the effect of dietary fish oil on gallbladder PG synthesis, and the relative abundance of various molecular species of phosphatidylcholines and phosphatidylethanolamines in bile and gallbladder epithelium in the cholesterol-fed prairie dog. Prairie dogs were maintained for 4 weeks on one of four diets: i) control, ii) cholesterol-supplemented (0.34%), iii) menhaden oil (50 g/kg chow), or iv) cholesterol plus menhaden oil. Supplementation with menhaden oil resulted in a replacement of arachidonic and linoleic acids with EPA and docosahexaenoic acids in the phospholipids of bile and gallbladder mucosa. In cholesterol-fed animals, supplementation with menhaden oil prevented increased gallbladder PG synthesis. Menhaden oil also reduced the incidence of cholesterol monohydrate crystals among cholesterol-fed animals (9/20 with cholesterol plus menhaden oil vs 21/22 with cholesterol alone), but the improvement could not clearly be attributed to decreased PG synthesis since supplementation with menhaden oil also increased the total phospholipid concentration in bile, and decreased the degree of cholesterol saturation. These results demonstrate that dietary supplementation with omega-3 fatty acids significantly influences biliary phospholipids, and decreases the incidence of cholesterol monohydrate crystal formation in this animal model.

Cholesterol crystal uptake and metabolism by P388D 1 macrophages

Cholesterol monohydrate crystals are frequently detected in intermediate and advanced atherosclerotic lesions. Little is known regarding mobilization of this molecular form of cholesterol into metabolically active pools. To study a potential mechanism for mobilization of crystalline cholesterol, we examined its uptake by a mouse macrophage cell line (P388D 1). Crystals were overlayered on a (P388D 1). cell monolayer maintained in a serum-free medium. Following incubation, the monolayer was washed, and the cells were harvested and analyzed for crystal internalization. By transmission electron microscopy, crystals were found intracellularly surrounded by a bilayer membrane. Analyses of the cellular cholesterol ester content by gas-liquid chromatography and esterification of [ 14C]cholesterol indicated the conversion of crystalline cholesterol to cholesterol esters. This pathway for solubilization of cholesterol crystals by macrophages could play an important role in the regression of atherosclerotic lesions.

Bovine gallbladder mucin promotes cholesterol crystal nucleation from cholesterol-transporting vesicles in supersaturated model bile.

This study examined the ability of purified gallbladder mucin to accelerate the nucleation of cholesterol monohydrate crystals from the cholesterol-transporting particles in supersaturated model bile. Mixed lipid micelles and cholesterol-phosphatidylcholine vesicles in supersaturated model bile were separated by Sephadex G-200 column chromatography. Mixed lipid micelles prepared by column chromatography had a low cholesterol-phosphatidylcholine ratio (0.30) and did not spontaneously nucleate cholesterol monohydrate crystals. In contrast, vesicles prepared by column chromatography had a cholesterol-phosphatidylcholine ratio of 1.00 and nucleated cholesterol crystals rapidly (P less than 0.001). Nucleation of cholesterol crystals was significantly accelerated in a concentration- and time-dependent manner by purified bovine gallbladder mucin in cholesterol containing vesicles, but not in mixed lipid micelles (P less than 0.001). A rapid filtration binding assay demonstrated significant binding of cholesterol and phosphatidylcholine in vesicles to gallbladder mucin but only minimal binding of cholesterol and phosphatidylcholine in mixed micelles. These data indicate that gallbladder mucin binds cholesterol and phosphatidylcholine in vesicles and accelerates the nucleation of cholesterol monohydrate crystals from these cholesterol-transporting particles in supersaturated model bile.

Association between cholesterol-phospholipid vesicles and cholesterol crystals in human gallbladder bile.

Rapid aggregation of cholesterol-phospholipid vesicles in gallbladder bile seems to be the first event in the production of cholesterol crystals, a prerequisite for cholesterol gallstone formation. We examined the amount of these vesicles in 33 human gallbladder biles in relation to biliary lipid composition and to the presence of cholesterol crystals. Biliary microscopy detected cholesterol crystals in all 19 biles from patients with cholesterol gallstones but in none of 14 biles from patients with pigment stones. Gel chromatography was used to separate vesicles and micelles in the native bile with an eluting buffer containing 10 mM sodium cholate to prevent disruption of micellar lipids. Cholesterol, phospholipid and bile salt concentrations were measured in every fraction collected. Bile acid, phospholipid, cholesterol and total lipid concentrations were not significantly different in samples with and without cholesterol crystals. The cholesterol saturation index (1.4 +/- 0.11 vs. 1.0 +/- 0.08) was significantly (p less than 0.01) higher in biles with crystals than without crystals. Gel filtration revealed a vesicular peak in addition to micellar fraction in 18 (23.1 +/- 3.2% of total cholesterol) of the 19 biles with crystals but only in three (15.7 +/- 2.4% of total cholesterol) of 14 biles without crystals. There was no relation between biliary lipid concentration or the cholesterol saturation index and the percentage of vesicular cholesterol in biles with or without crystals. The close association of vesicles and crystals in human gallbladder bile supports the contention that vesicles are important in the initial nucleation of cholesterol monohydrate crystals.

Effect of hyocholic acid on the prevention and dissolution of biliary cholesterol crystals in mice

Gallstone prevention and dissolution were studied in a mouse model of cholesterol cholelithiasis using hyocholic acid (3 alpha, 6 alpha, 7 alpha-trihydroxy-5 beta-cholanic acid). Addition of hyocholic acid, 0.1 or 0.3%, in the lithogenic diet (1% cholesterol + 0.5% cholic acid) prevented the formation of cholesterol monohydrate crystals in 70 and 90% of cases, respectively. On the other hand, chow diet supplemented with 0.1 or 0.3% hyocholic acid dissolved cholesterol crystals in lithiasic mice in, respectively, 80 and 100% of cases within 12 days. In both protocols, biles were largely supersaturated with cholesterol; lecithin-cholesterol lamellar liquid crystals were responsible for the transport of the excess cholesterol content. The percentage of hydrophilic bile salts (hyocholic acid, hyodeoxycholic acid, beta-muricholic acid) in bile, although moderate (15-50% of total bile salts), appears to induce such liquid crystalline dispersion. This study demonstrates that the balance between hydrophilic and hydrophobic bile salts plays a major role in the prevention and dissolution of cholesterol crystals. It is also shown that the desaturation of biliary cholesterol is not a prerequisite for gallstone dissolution.

Occurrence of cholesterol monohydrate crystals in gallbladder and hepatic bile in man: influence of bile acid treatment

The occurrence of cholesterol monohydrate crystals was examined and related to the degree of cholesterol saturation in gallbladder bile and hepatic bile of gallstone (GS) patients (n = 34), gallstone-free (GSF) subjects (n = 33) and GS patients treated with chenodeoxycholic acid (CDCA (n = 7) or ursodeoxycholic acid (UDCA) (n = 11) for 3 weeks prior to cholecystectomy. Twenty-five untreated GS patients (74%) and four UDCA-treated patients (40%) displayed cholesterol crystals in the gallbladder bile. Only two GSF subjects (6%) and none of the CDCA-treated patients had crystals. Half of the patients with crystals in the gallbladder bile had crystals also in the hepatic bile. Cholesterol saturation of the gallbladder bile was higher in GS (142 +/- 15%, mean +/- SEM) than in GSF patients (74 +/- 5%). Saturation was also higher in GS patients with crystals (157 +/- 20%) than in those without crystals (99 +/- 12%). Gallbladder bile was unsaturated in all CDCA- and UDCA-treated patients. The results underline the importance of the degree of cholesterol saturation for the formation of cholesterol crystals. The data also give further support to the concept that the mechanism for inducing gallstone dissolution is different for CDCA and UDCA.

The emergence of biliary sludge, a new clinical entity.

Biliary sludge is a collection of mucus, calcium bilirubinate, and cholesterol crystals that is usually recognized by characteristic echoes on ultrasonography. Its pathogenesis, clinical significance, and ultimate prognosis remain uncertain. We therefore studied the origin of biliary sludge ultrasonic echoes, using an ex vivo liver-gallbladder preparation, and determined the outcome of a group of patients identified to have gallbladder sludge by ultrasonography. Echoes were not generated by either an increase in the total solid concentration or by the graded addition of partially purified mucus glycoprotein. Cholesterol monohydrate crystals (>50 μm) mixed with mucus produced echoes that were indistinguishable from gallbladder sludge observed in patients. To determine the natural evolution of gallbladder sludge in patients, we prospectively followed 96 patients found to have biliary sludge for a mean of 37.8 mo by serial ultrasound scans every 6 mo. In 17 patients (17.7%) biliary sludge disappeared and did not recur for at least 2 years, in 58 patients (60.4%) biliary sludge disappeared and reappeared, and in 8 patients (8.3%) asymptomatic gallstones developed. There were 12 cholecystectomies performed: six were done for symptomatic gallstones (6.3%) and the other six for sludge associated with severe biliary pain attacks with or without recurrent acute pancreatitis. The finding of sludge represented precipitates being formed in bile. In some patients, it was a precursor form of gallstone disease.