Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones.

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.

Percutaneous pulsed-dye laser lithotripsy of gallbladder stones in swine.

Human gallstones were surgically implanted in the gallbladders of 14 pigs. Nine to 16 days later a sheath was successfully placed percutaneously into the gallbladders of 13 animals using ultrasound and fluoroscopy. Two methods were used to guide laser fragmentation: (1) fluoroscopy and a steerable double lumen catheter (two animals), and (2) a flexible endoscope (11 animals). Laser treatment was done in 12 animals with a flashlamp-pumped pulsed-dye laser. A mean of 3600 pulses/animal were delivered using a wavelength of 504 nm and a maximum energy of 60 mJ/pulse. No fragmentation occurred in two animals, partial fragmentation occurred in six, and complete fragmentation occurred in four. Endoscopic guidance was superior to fluoroscopic guidance. Complications (sheath dislodgment, gallbladder perforation, bleeding) occurred in eight of 14 animals. Pulsed-dye laser fragmentation of gallbladder stones is feasible using endoscopic guidance. The use of this technique through an acute percutaneous tract may be associated with complications.

Type of gallstones and deaths from stroke and coronary heart disease among cholecystectomized patients.

A total of 841 Japanese patients who had undergone cholecystectomy for choleithiasis (550 for cholesterol stones and 291 for pigment stones) from 1951 to 1970 were investigated on death from stroke, coronary heart disease (CHD) and heart disease other than CHD in relation to the type of gallstones. Compared to patients with pigment stones, those having cholesterol stones had a 50% lower risk of dying from stroke which was statistically significant. The findings support the idea that westernization of Japanese diets may be responsible both for the decline in stroke mortality and for the changing pattern of gallstones in this country. The risk of CHD among cholesterol-stone patients was higher, but not significantly so, than that of pigment-stone patients whereas mortality from heart disease other than CHD did not differ much between the two groups. There were, however, few deaths from these diseases and the findings were therefore not conclusive.

Gallstone disease. The clinical manifestations of infectious stones.

Gallstones from 82 patients were examined under a scanning electron microscope for evidence of bacteria, and the findings were compared with the clinical manifestations of the disease. Bacteria were present in 68% of pigment stones and the pigment portions of 80% of composite stones. These gallstones were referred to as infectious stones. No bacteria were found in cholesterol gallstones. Acute cholangitis was diagnosed in 52% of patients with infectious stones and in 18% of patients with noninfectious stones. Over half of the patients with noninfectious stones presented with mild symptoms. Infectious stones were more often associated with a previous common duct exploration, an urgent operation, infected bile, a common duct procedure, and complications. These data show that gallstone disease is more virulent in patients whose gallstones contain bacteria.

Reversal of pigment gallstone disease in a canine model.

Unlike dietary-induced cholesterol gallstones, which may disappear spontaneously when the lithogenic diet is withdrawn, little is known about the natural history of pigment gallstones. We examined whether pigment gallstone disease, which can be uniformly induced in the dog by six weeks of a methionine-deficient diet, can be reversed by return to normal diet. As previously reported, all dogs develop pigment gallstones as well as significant increases in biliary total calcium, free ionized calcium, and cholesterol concentrations after six weeks of a lithogenic diet. These changes are accompanied by a significant increase in the concentration of unconjugated bile salts in bile. In addition, histologic changes in the gallbladder wall occur that are consistent with a moderate degree of chronic cholecystitis. This study clearly demonstrates that return to a normal diet for six weeks allows bile composition to normalize, gallstones to disappear in 50% of dogs, and gallbladder histologic changes to return toward normal. Thus, it would appear that pigment gallstone disease in this model may be reversible, at least early during its course. Although the relevance of these findings to pigment gallstones in humans must be established, the potential for nonoperative treatment of pigment gallstones should not be discounted.

Risk factors for acute pancreatitis in patients with migrating gallstones.

Stool screening for gallstones and ultrasound monitoring of diameter changes of the biliary and pancreatic duct were performed in 129 patients with choledocholithiasis. Gallstone migration was found in 44 patients, all of whom were operated on electively. At surgery, acute pancreatic lesions were found in 16 patients; in the remaining 28 there was no evidence of pancreatic inflammation. There were no significant differences among patients in both groups regarding sex, age, stone size, shape or number found in stools, interval between admission and migration, or the presence of a dilated pancreatic duct before migration. Pancreatic duct reflux, however, was significantly more frequent in cholangiograms of patients with acute pancreatitis, implying that a common channel may be a major factor relating to acute pancreatitis in patients with migrating gallstones.

Gallstone lithotripsy by extracorporeal shock waves.

Extracorporeal shock-wave lithotripsy (ESWL) in combination with adjuvant litholytic therapy using chenodeoxycholic acid and ursodeoxycholic acid (7 to 8 mg/kg body weight/day of each acid) is a safe and effective, novel nonsurgical approach to gallbladder stones, provided the patients are carefully selected. Experience has shown that patients with a radiolucent solitary stone in a functioning gallbladder are the best candidates. In addition, ESWL is a worthwhile noninvasive alternative to open surgery in patients with bile duct stones in whom routine endoscopic measures have failed.

Dissolution of bile duct stones.

Mono-octanoin can be used either alone or as an adjunct to other techniques to dissolve cholesterol bile duct stones. This solvent can be administered through an existing T tube, through the nasobiliary route, or percutaneously through the liver. Unlike basket extraction, which requires a mature T-tube sinus tract, mono-octanoin can be used immediately postoperatively or for home dissolution therapy. The endoscopic extraction of bile duct stones has a 1 percent mortality rate and a 5 to 7 percent complication rate. Special mixtures of mono-octanoin, bile acids, and ethylene diaminetetraacetic acid (EDTA) are being evaluated to dissolve pigment stones. The use of methyl tert-butyl ether is still experimental but very effective. To be most successful, mono-octanoin treatment must be used in properly selected patients.

Increased biliary calcium in cholesterol gallstone formation.

The hypothesis that biliary calcium levels increase during cholesterol gallstone formation was tested in a prairie dog gallstone model. In addition to the previously documented changes in biliary lipid composition, animals with gallstones had a significant increase in gallbladder bile concentrations of total and ionized calcium compared with control animals. The observation that hepatic bile levels of calcium remained unchanged in the cholesterol-fed animals suggests that the increase in gallbladder bile calcium is due to a gallbladder-related phenomenon, rather than an alteration in hepatic metabolism. We proposed that this increase in biliary calcium levels results from an increase in gallbladder absorption. Increased biliary calcium levels may be an important, previously unrecognized, factor in the pathogenesis of cholesterol gallstones.

Cholelithiasis in the Syrian population. A prospective study of 189 patients.

A prospective study of 189 Syrian patients with cholelithiasis was performed to throw light on some features of the disease in Syria. Early or emergency cholecystectomies were performed in 28 percent of all operations, whereas elective operations were carried out in the remaining patients. Explorations of the common bile ducts were performed in 34 patients, only 4 of whom had no stones. Wound and chest infections represented the most common postoperative complications. There was only one postoperative death, which was reportedly due to massive hemorrhage from esophageal varices. Analysis of the stones removed after cholecystectomies revealed that 72.4 percent were of mixed composition, 19.4 percent were pure cholesterol, and 8.2 percent were pigmented stones.

Medical dissolution of gallstones by oral bile acid therapy.

The rationale, safety, and efficacy of cholesterol gallstone dissolution by orally administered ursodiol, chenodiol, or a combination of the two agents are summarized herein. Bile must be supersaturated in cholesterol for gallstones to form, and desaturation of bile by orally administered bile acids induces gradual stone dissolution. The mechanism of action of the two agents differs, but both cause a decreased input of cholesterol into the metabolic pool. Ursodiol is free of side effects, and the combination with chenodiol is equally efficacious and also has few side effects. Chenodiol, although an effective desaturation agent, causes diarrhea, mild reversible hepatic injury, and a small increase in the plasma cholesterol level. Extracorporeal shock-wave lithotripsy decreases gallstone size markedly and thereby increases the speed of dissolution by orally administered bile acids. Medical therapy with oral bile acids is appropriate for patients who present with small cholesterol stones and for patients with larger cholesterol gallstones who cannot or will not have surgery. Oral bile acids may also be valuable in the treatment of gallstone recurrence before it has become symptomatic or to prevent recurrence after prior successful dissolution of recurrent stones.

Structural and chemical characterization of gallstones resistant to dissolution therapy.

X-ray diffraction, i.r. spectroscopic, and chemical analyses have been carried out on radiolucent gallstones resistant to dissolution therapy. Cholesterol represents the main component of all the examined stones, while the ratio between the amounts of pigmented material and calcium carbonate is about 1 in the inner and outer layers of the stones and 3 in the medial layer. Calcium carbonate is present in two distinct crystalline forms: vaterite, which is the main inorganic crystalline phase, and calcite. The cell parameters of vaterite and calcite are shorter in the inner and outer layers of the stones than in the medial layer. The observed variation of the cell parameters has been related to the substitution of copper to calcium in the carbonate structures, on the basis of the data obtained on vaterite and calcite synthesized in presence of different copper concentrations in solution. The results indicate that the failure of the dissolution therapy can be related to the inhomogeneous distribution in the stones of calcium carbonate and calcium bilirubinate.

XeCl laser in biliary calculus fragmentation: fluence threshold and ablation products.

The in vitro action of a xenon-chlorine (XeCl) excimer laser on biliary calculi is reported: fluence threshold and rate for ablation process are given. An analysis of gaseous products evolved during irradiation of gallstones, performed through an infrared spectrophotometric technique is also reported. Based on the different results, we discuss the mechanism of destruction.

Cholesterol gallstone disease: the current status of nonsurgical therapy.

Gallstone disease is a common disease that appears to be related to a Western diet. The underlying pathogenesis is a subtle alteration in the liver such that excessive cholesterol is extracted from the liver cell by bile acids undergoing an enterohepatic recirculation. Gallstone disease progresses through well-defined stages, beginning with a bile supersaturated with cholesterol and proceeding to crystal formation, stone growth, and finally symptoms caused by impaction of a stone in either the cystic duct or the common bile duct. The natural history is that most stones never cause symptoms. Stones that cause symptoms have been present for an average of 12 years. The treatment of truly asymptomatic stones should be observation. Ultrasonography of the right upper quadrant is the gold standard for the diagnosis of stones in the gallbladder. Endoscopic retrograde cholangiopancreatography (ERCP) is the gold standard for the diagnosis of stones in the common bile duct. Oral cholecystogram (OCG) helps select patients who have noncalcified, floating stones that may be dissolved with bile acids or methyl tertiary butyl ether (MTBE). Therapy with chenodiol has been a disappointment because of a low complete response rate. The ideal candidate for attempted dissolution with chenodiol would be a thin woman with hypercholesterolemia and a small number of symptomatic, small, floating, radiolucent gallstones. Ursodeoxycholic acid (Urso), when it is available, will have all of the attributes of chenodiol and virtually none of the side effects. Rapid dissolution of gallstones with MTBE shows great promise of being a generally available means of dissolving gallstones. Extracorporeal shock wave lithotripsy also shows promise, but its general availability may be limited by the cost of the equipment needed. As of now, the treatment of choice for symptomatic gallstones remains cholecystectomy, unless there is a compelling reason not to operate.

Effect of previous nutritional status on the formation of cholesterol gallstones in the prairie dog.

In the prairie dog model of cholesterol cholelithiasis, a high incidence of gallstones is achieved by feeding a semipurified lithogenic diet containing 0.4% cholesterol for 2 mo. On occasion, we noted a decrease in the percentage of animals with gallstones from 90-100% to 50-55%. To explain this phenomenon, we studied the effect of dietary history on gallstone formation. After weaning, animals were fed either rodent chow or alfalfa plus corn (mo 0-3) followed by a cross-over experiment at mo 4-6. Gallstone formation then was studied by feeding the lithogenic diet from mo 7 to 8. At sacrifice, the incidences of gallstones, biliary lipids and tissue cholesterol levels were correlated with dietary history. The incidence of gallstones was 100% only in animals fed the alfalfa-corn diet from weaning to 3 mo. In addition, the feeding of the alfalfa-corn diet at mo 4-6 increased gallstone incidence from 65% to 86%. The lithogenic index of all groups was highest when the animals received only alfalfa-corn prior to the lithogenic stimulus. The activity of hepatic HMG-CoA reductase was elevated in animals fed alfalfa-corn from weaning to 8 mo, suggesting that this diet stimulates hepatic cholesterol synthesis, leading to increased biliary cholesterol secretion. It is concluded that previous nutritional conditioning affects the incidence of gallstones. The prairie dog is a useful model of cholesterol cholelithiasis, but the dietary history of the animals plays an important role in lithogenesis.

The effect of alfalfa-corn diets on cholesterol metabolism and gallstones in prairie dogs.

Cholesterol gallstones were present in prairie dogs fed alfalfa plus corn with and without exogenous cholesterol (0.4%). The diets fed to the animals for eight weeks contained alfalfa plus corn in fixed proportions of 50:50, 85:15 and 15:85 (w/w). At sacrifice, all animals were healthy but had not gained weight; no deaths occurred during the experiment. Cholesterol gallstones were present in all groups. In the absence of exogenous cholesterol, the highest stone incidence was found in the animals which received the lowest fiber (highest corn) diets (alfalfa plus corn, 50:50, 67%; alfalfa plus corn, 15:85, 83%). Cholesterol gallstone incidence was 100% when exogenous cholesterol was added to the alfalfa plus corn diets (50:50 and 15:85). No pigment gallstones were detected in any animal. Liver and plasma cholesterol concentrations were highest in the animals receiving alfalfa plus corn (15:85) plus 0.4% cholesterol (4.29 mg/g, and 356 mg/dl, respectively). These values were lowest in animals receiving 85% alfalfa plus 15% corn without cholesterol (2.19 mg/g and 88 mg/dl, respectively). Lithogenic indices were below 1.00 in all groups. Biliary bile acids were mainly amidates of cholic acid and chenodeoxycholic acid, with the former predominating. Thus, gallstones can be formed in prairie dogs in the absence of exogenous cholesterol; gallstone incidence is reduced by dietary fiber.

Cholelithiasis in hamsters: effects of cholic acid and calcium on gallstone formation.

Dietary cholic acid (0.1%) and/or calcium (2.6% as calcium carbonate) were added to a semipurified diet containing cholesterol and ethynyl estradiol to determine whether the incidence of pigment and/or cholesterol gallstones would be changed. Male golden Syrian hamsters were fed the experimental diets for 96 days (Group 1, control; Group 3, cholic acid plus calcium) or only an average of 60 days (Group 2, 0.1% cholic acid). Animals in Group 2 became ill (weight loss, low food intake, diarrhea) possibly due to cholic acid (or deoxycholic acid) toxicity. Cholesterol gallstones and crystals were absent in all experimental groups. The incidence of pigment gallstones was: control, Group 1, 12/16; 0.1% cholic acid, Group 2, 3/13; and 0.1% cholic acid plus calcium, Group 3, 11/22. Cholic acid with or without calcium produced an elevation of both liver and plasma cholesterol: Group 2, 80.1 mg/g and 501 mg/dl; Group 3, 103.7 mg/g and 475 mg/dl vs Group 1, 65 mg/g and 209 mg/dl, respectively. The lithogenic indices of the bile were lower in Groups 2 and 3 compared to Group 1, controls, 0.45 and 0.58 vs 1.16, respectively. The extent of the portal tract pathology could not be correlated with the presence or absence of pigment gallstones or with the levels of lithocholic acid in the hamster bile. In summary, when semipurified diets were supplemented with ethynyl estradiol and cholic acid, with and without calcium supplementation, no cholesterol gallstones formed and the incidence of pigment gallstones was not altered.

Pigment vs. cholesterol microlithiasis: comparison of clinical features, bacteriology, stone and gallbladder composition.

We compared 16 cases of pigment microlithiasis with 12 cases of cholesterol microlithiasis. In each case we made a spectrophotometric and diffractometric analysis of the composition of the stones, also analysing bile bacteriology, bile lipid composition and cholesterol saturation indices. The two groups were comparable as regards sex, age, symptoms and clinical features. Pigment microcalculi were often asymptomatic (41.7%), but more often associated with bile bacteria (43.7%) or acute pancreatitis (25%). The results were such as to permit us to view cholesterol microlithiasis and pigment microlithiasis as having different pathogenetic and clinical aspects, and thus as being different diseases.

Spontaneous cholelithiasis in sheep: prevalence survey and analysis of gallstones and bile.

In an abattoir survey of the consecutive series of 206 sheep at a local slaughterhouse, gallstones (concretions with a diameter equal to or greater than 1 mm) were found in the gall bladder of 24 animals. There were stones in 19 out of 146 adult sheep and in 5 out of 60 lambs, with the higher frequency in the male sex in both groups. Qualitative analysis of the stones showed that all of them were pigment (bilirubin) stones. There was no statistically significant difference (paired t test, P greater than 0.05) between the amounts of biliary cholesterol, phospholipids, bile acids and total and indirect bilirubin in 12 sheep with gallstones and 12 control sheep without gallstones, paired for age, sex and day of slaughter.