Pigment vs cholesterol cholelithiasis

Although pigment (calcium bilirubinate) gallstones in Japanese subjects are associated with bacterial infection, the role of infection in Americans with pigment gallstones has not been assessed. Anaerobic and aerobic cultures of gallbladder bile, stone, and wall were obtained at cholecystectomy from nine patients with pigment stones and 25 with cholesterol stones. Among pigment-stone subjects, only 1 of 9 grew organisms in greater than 10⁵ colony-forming units (CFU)/ml or g in gallbladder bile or wall. Likewise, growth greater than 10⁵ CFU/ml or g was present in only 1 of 26 biles and 2 of 26 walls from cholesterol-stone patients.Propionibacterium acnes was found in less than 10⁵ CFU/g or ml in at least 1 specimen from 6 of 9 pigment- and 12 of 26 cholesterol-stone patients. This organism was considered a contaminant because propionic acid concentrations in bile, an index of active bacterial metabolism, were similar in specimens with or without low-titer growth. The concentrations of bile salts, phospholipids, cholesterol, and bilirubin in gall-bladder bile was unaffected by the type of bacteria in low-titer growth. But the lipid concentrations were markedly depressed in two biles with bacterial growth greater than 10⁵ CFU/ml. The molar ratio of bile salts and phospholipids to cholesterol was significantly higher in biles surrounding pigment stones than those surrounding cholesterol stones (P<0.01). We conclude that significant bacterial infection (>10⁵ CFU/ml) is not associated with pigment or cholesterol stones in asymptomatic American subjects at cholecystectomy. These data suggest that pigment-stone formation in the United States is not primarily related to bacterial alteration of bile composition, as the experience with Japanese patients would suggest.Supported by NIH Grant AM-16549 and institutional funds of the Veterans Administration.Presented in part at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, May 8–13, 1977.     

Gallstone formation and gallbladder bile composition after colectomy in dogs

A high prevalence of gallstones has been described in patients following colectomy. The aim of this study was to examine whether lithogenicity is attributed to colectomy. In the present study, changes in gallbladder bile composition and the mechanism of gallstone formation after colectomy were examined in dogs. Ten mongrel dogs underwent restorative proctocolectomy. Seven dogs which received sham operations served as controls. Over a 12-week postoperative period, samples of gallbladder bile, formed gallstones and serum were collected and analyzed. In 7 of the 10 (70%) colectomized dogs, gallstones were found in the gallbladder, while the control dogs had no stones. Macroscopically the gallstones were similar to black pigment stones observed in humans. Chemical analysis and Fourier transform-infrared spectroscopy examination revealed that the stones were composed mainly of sodium bilirubinate and proteins, with minor amounts of calcium salts and cholesterol. Significant increases in biliary pH and concentrations of ionized calcium and unconjugated bilirubin were observed in the gallbladder bile of the colectomy group compared with that of the control group. The total bile acid and total bilirubin concentrations were significantly decreased in the colectomy group. Cholesterol crystal nucleation did not occur. The inhibitory effect of gallbladder bile on calcium carbonate precipitation in anin vitro assay system was preserved even after colectomy. In conclusion, proctocolectomy increases the concentration of unconjugated bilirubin in gallbladder bile and induces pigment gallstones which are composed mainly of sodium bilirubinate and proteins since calcium ions and cholesterol are stabilized in dogs.     

Physical and metabolic factors in gallstone pathogenesis

Gallstones form when the tenuous balance of solubility of biliary lipids tips in favor of precipitation of cholesterol, unconjugated bilirubin, or bacterial degradation products of biliary lipids. For cholesterol gallstones, metabolic alterations in hepatic cholesterol secretion combine with changes in gallbladder motility and intestinal bacterial degradation of bile salts to destabilize cholesterol carriers in bile and produce cholesterol crystals. For black pigment gallstones, changes in heme metabolism or bilirubin absorption lead to increased bilirubin concentrations and precipitation of calcium bilirubinate. In contrast, mechanical obstruction of the biliary tract is the major factor leading to bacterial degradation and precipitation of biliary lipids in brown pigment stones. Further understanding of the physical and metabolic factors of cholesterol and black pigment formation is likely to provide interventions to interrupt the earliest stages of gallstone formation.     

Cholelithiasis in Taiwan

The nature and occurrence of gallstones in Taiwan and their etiologic factors might not be the same as in Western countries and warranted a systematic investigation. Gallbladder biles and gallstones were obtained at surgery from 100 and 74 patients, respectively. Common duct bile and stones were either drained through an indwelling common duct T-tube or aspirated through a nasobiliary catheter in 108 patients. Gallstones were analyzed for bilirubin, cholesterol, bile acid, calcium, and residue, and biles for bile acid, cholesterol, phospholipid, bilirubin, and β-glucuronidase. There were four major kinds of gallstones in Taiwan: cholesterol/mixed stones, high-residue black formed pigment stones, low-residue brown formed pigment stones, and muddy pigment stones. The surgical incidence of all types of stones increased steadily during the past four decades. During the past 15 years the relative frequencies for mixed, formed pigment, and muddy pigment stones had been roughly 40, 40, and 20%, respectively, with a further increase in the mixed stones and a decrease in the muddy pigment stones in recent years. Improvement of nutritional status and living standards might contribute to such changes. Cholesterol content in the common duct and gallbladder biles was higher in the mixed stone group than in other groups. Bacterial β-glucuronidase activity was detected in 53% of patients with muddy pigment stones. Endogenous β-glucuronidase activity and concentration were also highest in this group, intermediate in the formed pigment and mixed stone group, and lowest in the control. We concluded that hypercholesterobilia was responsible for increasing incidence of mixed stones during the past two decades, while both bacterial and human β-glucuronidase might contribute to pigment cholelithiasis.     

Structure and composition of primary intrahepatic stones in Korean patients

We have analyzed the chemical composition of primary intrahepatic stones from 72 Korean patients. Two types of concretions have been identified: brown pigment (calcium bilirubinate) stones and black-colored mixed stones. Brown pigment stones were found in 68% of all cases and the remainder (32%) consisted of mixed stones. Intrahepatic mixed stones had mean cholesterol and bilirubin contents of 46.6% and 25.9%, respectively, whereas calcium bilirubinate stones had mean cholesterol and bilirubin contents of 14.1% and 43.6%, respectively. Intrahepatic mixed stones had a smooth black-colored surface and on cross section, exhibited a distinct outer shell surrounding an inner yellow, cholesterol-rich body. The finding of intrahepatic mixed stones with high cholesterol content suggests that primary hepatolithiasis may result from at least two different conditions or disorders and points to different approaches to their treatment.     

Cholesterol and pigment gallstone disease: comparison of the reliability of three bile tests for differentiation between the two stone types

Gallbladder biles and stones were obtained at 116 cholecystectomies for symptomatic gallstone disease. All 33 patients younger than 50 years had cholesterol stones, whereas 40% of the older patients had pigment stones. We compared the reliability of three different bile tests for the differentiation between cholesterol and pigment stone patients. Whereas both the presence of cholesterol monohydrate crystals in fresh gallbladder bile and a nucleation time less than or equal to 20 days in ultrafiltered gallbladder bile had a specificity of 100% for cholesterol gallstone disease, biliary supersaturation with cholesterol (cholesterol saturation index greater than 1.0) had a low specificity. The sensitivity of nucleation time less than or equal to 20 days for cholesterol gallstone disease was 78% in concentrated gallbladder biles (biliary total lipid concentration greater than or equal to 5 g/dl) but only 21% in dilute biles (biliary total lipid concentration less than 5 g/dl). In contrast, examination for the presence of cholesterol crystals in fresh bile was reasonably sensitive both in concentrated and dilute gallbladder biles (sensitivity, 84% and 72%, respectively). In addition, duodenal bile obtained from 16 patients (10 cholesterol, 6 pigment) before cholecystectomy showed cholesterol crystals in 7 of the cholesterol but in none of the pigment stone patients. We conclude that examination of fresh bile for cholesterol crystals is a specific and reasonably sensitive test for cholesterol gallstone disease.     

The usefulness of microscopic bile examination in patients with suspected microlithiasis: a prospective evaluation

Gallbladder bile collected by duodenal intubation or during surgery was examined microscopically in patients who were free of stones and in patients with proven stones. None of the 16 patients free of stones had cholesterol monohydrate crystals or calcium bilirubinate granules in bile. Among the 17 patients with proven cholelithiasis, 13 with cholesterol stones had cholesterol monohydrate crystals in their bile, but only 2 of the 4 patients with pigment stones had calcium bilirubinate granules. These data confirm that cholesterol monohydrate crystals are sensitive and specific for cholesterol stones, whereas calcium bilirubinate granules lack sensitivity for the diagnosis of pigment stones. From these results, the diagnostic usefulness of microscopic examination of bile collected from the duodenum was studied prospectively in 46 patients with symptoms suggestive of cholelithiasis but in whom stones had not been visualized at cholecystography and ultrasonography. In 15 of them, bile was found to be abnormal: cholesterol monohydrate crystals were seen in 11, cholesterol crystals + calcium bilirubinate granules in 2 and calcium bilirubinate granules in 2. To date, nine of these patients have been operated on: 6 (all with cholesterol monohydrate crystals) had small cholesterol gallstones and 3 (2 with cholesterol monohydrate crystals and 1 with calcium bilirubinate granules) had signs strongly suggestive of the recent migration of gallstones. One patient refused operation, but minute pigment stones were found to be associated with calcium bilirubinate granules at duodenal intubation. In the other 31 patients, bile contained neither cholesterol monohydrate crystals nor calcium bilirubinate granules. They were not operated on and were followed up with repeated investigations for 12 to 24 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a concanavalin A-binding biliary glycoprotein on nucleation time of gallbladder bile

A study was performed to determine quantitative differences in the total protein concentration of gallbladder bile from gallstone patients and to isolate nucleation-promoting factors from the bile. Total protein concentrations in cholesterol gallstone bile (3.6±0.6 mg/ml, mean±SD, n=10), calcium bilirubinate gallstone bile (4.2±1.1 mg/ml, n=10), black pigment gallstone bile (1.9±0.6 mg/ml, n=4) and control gallbladder bile (2.3±0.5 mg/ml, n=9) were not significantly different. Also no statistically significant differences in cholesterol saturation index were found among these groups. Gallbladder bile from cholesterol gallstone patients showed significantly faster nucleation than that of contorls, calcium bilirubinate gallstone, or black pigment gallstone patients. We partially purified biliary glycoproteins from cholesterol gallstone bile or calcium bilirubinate gallstone bile by chromatography on concanavalin A Sepharose. Nucleation time was measured following the addition of these proteins to control bile in vitro. The glycoproteins obtained from cholesterol gallstone bile had significant nucleation-promoting activity, but nucleation time was not changed following the addition of biliary glycoproteins from calcium bilirubinate gallstone patients. These results suggest that qualitative differences in individual proteins of gallbladder bile are responsible for nucleation-promoting activity in vitro.     

Effects of a concanavalin A-binding biliary glycoprotein on nucleation time of gallbladder bile

A study was performed to determine quantitative differences in the total protein concentration of gallbladder bile from gallstone patients and to isolate nucleation-promoting factors from the bile. Total protein concentrations in cholesterol gallstone bile (3.6 +/- 0.6 mg/ml, mean +/- SD, n = 10), calcium bilirubinate gallstone bile (4.2 +/- 1.1 mg/ml, n = 10), black pigment gallstone bile (1.9 +/- 0.6 mg/ml, n = 4) and control gallbladder bile (2.3 +/- 0.5 mg/ml, n = 9) were not significantly different. Also no statistically significant differences in cholesterol saturation index were found among these groups. Gallbladder bile from cholesterol gallstone patients showed significantly faster nucleation than that of controls, calcium bilirubinate gallstone, or black pigment gallstone patients. We partially purified biliary glycoproteins proteins from cholesterol gallstone bile or calcium bilirubinate gallstone bile by chromatography on concanavalin A Sepharose. Nucleation time was measured following the addition of these proteins to control bile in vitro. The glycoproteins obtained from cholesterol gallstone bile had significant nucleation-promoting activity, but nucleation time was not changed following the addition of biliary glycoproteins from calcium bilirubinate gallstone patients. These results suggest that qualitative differences in individual proteins of gallbladder bile are responsible for nucleation-promoting activity in vitro.     

Composition of pigmented centers of cholesterol gallstones

Most cholesterol gallstones have visually pigmented centers, but it is unclear whether this represents simple co-precipitation of pigment with cholesterol during stone nidation or nidation on a true pigment stone center. To clarify this issue, we selected from among 67 sets of cholesterol gallstones, 12 sets with the most conspicuously pigmented centers. The composition of the centers and the peripheries of these 12 stones was analyzed using infrared spectroscopy and compared with that of 10 black pigment gallstones. The pigmented centers of cholesterol stones contained 80.1 +/- 7.9% (mean +/- S.E.) cholesterol, 6.2 +/- 3.4% calcium bilirubinate (only 4 of the 12 centers had measurable calcium bilirubinate), trace amounts of calcium phosphate and no calcium carbonate or calcium palmitate. The peripheral areas of the cholesterol stones contained 91.6 +/- 2.3% cholesterol and no detectable calcium salts. For comparison, the composition of the centers of 10 black pigment gallstones was 13.5 +/- 2.2% cholesterol, 28.2 +/- 5.3% calcium bilirubinate, 5.5 +/- 2.4% calcium phosphate and 10.6 +/- 5.8% calcium carbonate. The composition of only one cholesterol stone center (15.8% cholesterol, 26.8% calcium bilirubinate) resembled that of a pigment stone, but even this center differed from that of a typical pigment stone in that it contained only a trace amount of calcium phosphate and no calcium carbonate. Thus, the chemical composition of pigmented centers of cholesterol gallstones is quantitatively different from that of black pigment stones, suggesting that cholesterol gallstones do not form on a pigment stone nidus.     

Black and brown pigment gallstones differ in microstructure and microcomposition

The two subtypes of pigment gallstones, black and brown stones, differ in chemical composition and pathogenesis. We examined a black bilirubinate stone and a black phosphate stone (which represented opposite ends of the compositional spectrum of black noncarbonate stones), a black carbonate stone, and a brown pigment stone using scanning electron microscopy and microchemical techniques to determine if stone microstructure and microcomposition reflected different patterns of formation. The cross-sectional surfaces of the black bilirubinate and black phosphate stones were smooth and homogenous. Electron probe microanalysis demonstrated high concentrations of sulfur and copper in the center of the black bilirubinate stone; sulfur was in a low valence state consistent with disulfide linkages in proteins. The brown stone was rough-surfaced with lamellated bands on cross-section. The lighter-colored bands in this stone contained virtually all of the detected calcium palmitate, while the darker sections contained much more calcium bilirubinate. Plasma oxygen etching demonstrated a network of protein interdigitating with calcium bilirubinate salts in the black bilirubinate and black phosphate stones but not in the black carbonate or brown stones. Argon ion etching demonstrated that calcium bilirubinate was in a closely packed rod-shaped arrangement in all three black stones but not in the brown stone. We conclude that the marked differences in structure and composition between the black noncarbonate and brown pigment gallstones support the hypothesis that the two major pigment gallstone types form by different mechanisms. In addition, the layered structures of the black carbonate and brown stones suggest that stone growth is affected by cyclic changes in biliary composition.     

Secretion of biliary calcium is increased in dogs with pigment gallstones

We have previously demonstrated that gallbladder bile is supersaturated with calcium bilirubinate in a canine dietary model of pigment gallstones. Supersaturation resulted from combined increases in the concentrations of both biliary calcium and unconjugated bilirubin. The elevations in biliary calcium and unconjugated bilirubin concentrations remain unexplained but could possibly be due to increases in hepatic or ductular secretion, alterations in bile composition with respect to calcium- or bilirubin-binding affinity, decreases in absorption from the gallbladder lumen, or, in the case of unconjugated bilirubin, production within the lumen by hydrolysis of conjugated bilirubin. Here, we study a single possible cause for the observed increase in biliary calcium concentration during pigment gallstone formation in dogs. Secretion of calcium into bile in dogs with pigment gallstones before and after infusion of the bile salt, taurocholate, was compared to normal dogs. A significant increase in bile acid-independent bile flow and calcium output (CaO) was observed at any given bile acid output. Thus, plots of bile flow and CaO versus bile acid output yielded two separate functions in normal dogs and dogs with pigment gallstones. The slopes of these functions were similar, but intercepts extrapolated to zero bile acid output were markedly different, indicating that bile acid-independent, but not bile acid-dependent, bile flow and CaO was increased. The increase in CaO was not due to secretion of bile with increased concentrations of calcium but rather to the increases in the rate of bile flow. These findings might, in part, explain elevated calcium concentrations since increased amounts of calcium would be presented to the gallbladder in these animals during gallstone formation.This work was supported by a Veteran's Administration Merit Review Award and by NIH Grant 32130, National Institute of Diabetes, Digestive, and Kidney Diseases.     

Brown pigment stones in the common bile duct: reduced bilirubinate diconjugate in bile

BACKGROUND: Bilirubin is the main component of most common bile duct stones. Normally, almost all bilirubin in bile is conjugated to glucuronic acid or some other sugar moiety. These conjugates are unstable and liable to deconjugation. Unconjugated bilirubin is insoluble and may precipitate as the calcium salt found in brown pigment stones. The pattern of bilirubin conjugates in common duct bile of patients with choledocholithiasis has been unknown. METHODS: In a clinical series of 55 patients with choledocholithiasis common-duct bile was aspirated, and the bilirubin conjugates analyzed with high-performance liquid chromatography. One stone from each patient was analyzed for cholesterol and bilirubin content to determine stone type. RESULTS: Sixteen patients had cholesterol stones, 38 patients had brown pigment stones, and 1 patient had a black stone. Patients with pigment stones had a lower percentage of bilirubin diglucuronide (median, 60.3%; interquartile range, 49.7%-67.3%) than patients with cholesterol stones (64.0%; 60.2%-73.3%) (Mann-Whitney, P=0.015). No significant difference was found for the other bilirubin conjugates, total bilirubin, or biliary pH when pigment and cholesterol stone patients were compared. The time of bile sampling in relation to papillotomy and treatment of cholestasis was not associated with the low percentage of bilirubin diglucuronide. The observation of reduced values for bilirubin diglucuronide could not be ascribed to duodenal diverticula or Billroth-II gastric resection. CONCLUSION: The percentage of the main bilirubinate conjugate, bilirubin diglucuronide, is decreased in the common duct bile of patients with pigmented compared with cholesterol stones.     

Brown Pigment Stones in the Common Bile Duct: Reduced Bilirubinate Diconjugate in Bile

Background: Bilirubin is the main component of most common bile duct stones. Normally, almost all bilirubin in bile is conjugated to glucuronic acid or some other sugar moiety. These conjugates are unstable and liable to deconjugation. Unconjugated bilirubin is insoluble and may precipitate as the calcium salt found in brown pigment stones. The pattern of bilirubin conjugates in common duct bile of patients with choledocholithiasis has been unknown. Methods: In a clinical series of 55 patients with choledocholithiasis common-duct bile was aspirated, and the bilirubin conjugates analyzed with high-performance liquid chromatography. One stone from each patient was analyzed for cholesterol and bilirubin content to determine stone type. Results: Sixteen patients had cholesterol stones, 38 patients had brown pigment stones, and 1 patient had a black stone. Patients with pigment stones had a lower percentage of bilirubin diglucuronide (median, 60.3%; interquartile range, 49.7%-67.3%) than patients with cholesterol stones (64.0%; 60.2%-73.3%) (Mann-Whitney, P = 0.015). No significant difference was found for the other bilirubin conjugates, total bilirubin, or biliary pH when pigment and cholesterol stone patients were compared. The time of bile sampling in relation to papillotomy and treatment of cholestasis was not associated with the low percentage of bilirubin diglucuronide. The observation of reduced values for bilirubin diglucuronide could not be ascribed to duodenal diverticula or Billroth-II gastric resection. Conclusion: The percentage of the main bilirubinate conjugate, bilirubin diglucuronide, is decreased in the common duct bile of patients with pigmented compared with cholesterol stones.     

Studies on the pathogenesis of diet-induced dog gallstones

Experimental diet-induced dog gallstones contained mainly protein, mucous substances, bile salts, bilirubin, an insoluble pigment which formed an insoluble black residue after acid hydrolysis, and only traces of cholesterol. Added dietary cholesterol was necessary to pigmented gallstone production and led to hypercholesterolemia. In bile, the ratio of cholesterol to bile salts was increased, but phospholipids were increased and cholesterol insolubility was not found. Dry weight, osmolality, and concentration of sodium and potassium in bile were reduced, but were not considered sufficient to influence micelle formation or lipid-pigment solubility. Taurine was reduced in serum and bile and unconjugated bile acids appeared in gallbladder bile; the pKa of these acids is near the pH of bile in these animals and may have caused precipitation of bile acids, accounting for their presence in the stones. Bile cultures were sterile. Total bilirubin content was unaltered but the methods used did not exclude the presence of unconjugated bilirubin as a potential cause of pigment precipitation in aqueous bile. Increased numbers of secretory vesicles occurred in gallbladder epithelium and large amounts of mucus were in the epithelial crypts. These observations suggest that bile proteins or mucous substances are important to lithogenesis in this model.With the Technical Assistance of P.N. SineSupported in part by Graduate Training Grant in Gastroenterology 5T01-AM5206 and Research Grant AM 08708 from the National Institutes of Health, U.S. Public Health Grant Service, Bethesda, Maryland, and in part by a Veterans Administration Research and Education Associateship Award, and by the Medical Research Service.Dr. Freston is a Burroughs-Wellcom Scholar in Clinical Pharmacology     

Correlation of cholesterol and bilirubin solubilization in bile salt solution.

Not only cholesterol but also bilirubin were considered to be solubilized by bile salt micelles. The correlation of cholesterol and bilirubin solubilization in aqueous conjugated and unconjugated bile salts solution and the effect of calcium on their solubilization were studied in this report. Cholesterol solubilization was usually reduced to some extent with increasing amount of added bilirubin. Bilirubin solubilization was always reduced by the co-existence of solubilized cholesterol. It was found that the addition of calcium increased cholesterol solubilization in conjugated bile salts system. On the other hand, calcium reduced bilirubin solubilization due to the formation of insoluble calcium bilirubinate especially in a high pH range of unconjugated bile salts system. Cholesterol solubilization in conjugated bile salts system was relatively lower than unconjugated bile salts system with or without added calcium, however co-existing bilirubin minimized these differences. The pH-dependency of cholesterol and bilirubin solubilization was small in conjugated bile salts system. On the contrary, it was remarkably bigger in unconjugated bile salts-calcium system.     

Opisthorchiasis-associated biliary stones： Light and scanning electron microscopic study

AIM: Biliary stones are frequently encountered in areas endemic for opisthorchiasis in Thailand. The present study was to describe the prevalence and pathogenesis of these stones. METHODS: Gallstones and/or common bile duct stones and bile specimens from 113 consecutive cholecystectomies were included. Bile samples, including sludge and/or microcalculi, were examined for Opisthorchis viverrini eggs, calcium and bilirubin. The stones were also processed for scanning electron microscopic (SEM) study. RESULTS: Of the 113 cases, 82 had pigment stones, while one had cholesterol stones. The other 30 cases had no stones. Most of the stone cases (76%, 63/83) had multiple stones, while the remainder had a single stone. Stones were more frequently observed in females. Bile examination was positive for O. viverrini eggs in 50% of the cases studied. Aggregates of calcium bilirubinate precipitates were observed in all cases with sludge. Deposition of calcium bilirubinate on the eggshell was visualized by special staining. A SEM study demonstrated the presence of the parasite eggs in the stones. Numerous crystals, morphologically consistent with calcium derivatives and cholesterol precipitates, were seen. CONCLUSION: Northeast Thailand has a high prevalence of pigment stones, as observed at the cholecystectomy, and liver fluke infestation seems involved in the pathogenesis of stone formation.     

Spontaneous formation of pigmentary precipitates in bile salt-depleted rat bile and its prevention by micelle-forming bile salts

During studies on the effect of bile salt-pool depletion in the bile-fistula rat (adult male Sprague-Dawley), the spontaneous formation of an orange-brown precipitate was noted. The nature of this phenomenon and its relationship to BS and calcium concentration was investigated in depth. Bile from 18 animals was collected in the dark into transparent tubes containing sodium azide, ascorbic acid, and glucaro-1,4-lactone. The tubes were flushed with nitrogen, sealed, and incubated at 37 degrees C. The pigmentary precipitate formed in all the bile salt-depleted (less than 3-5 mM) bile samples (i.e., those collected after 5-7 h of external biliary drainage), but not in bile salt-rich biles. It appeared within 30-240 min after collection, both in bile samples collected at room temperature and at 37 degrees C, initially as a pale flocculation and then slowly sedimenting to form, after centrifugation, a solid, dark-orange pellet. There were no pH changes during incubation, and bile cultures were negative. Under polarizing microscopy, the precipitate appeared amorphous, and there was no evidence of birefringence. High-performance liquid chromatography showed that unconjugated bilirubin was the prevalent pigmentary component, but significant amounts of monoconjugated bilirubin also coprecipitated. Lipid chemistry showed the presence of lecithin (80.1% of total lipids), which was rich in palmitoyl and linoleoyl fatty acids, and of fatty acids (predominantly palmitic and oleic). Infrared spectroscopy and x-ray diffraction showed the presence of calcium bilirubinate and palmitate. In-vivo replenishment of the bile salt pool by intravenous infusion of either taurocholate or taurochenodeoxycholate (1 mumol/min) completely prevented the pigmentary precipitation. In vitro experiments showed inhibition of the precipitate formation by the addition of individual bile salt in concentrations approximating their critical micellar concentration. Precipitate formation was hastened by the addition of calcium chloride (4-12 mM), but only in bile salt-depleted biles. As the composition of the precipitate closely resembles that of human brown-pigment stones and sludge, these findings may provide new insights into an understanding of the pathogenesis of pigment gallstone disease.     

Pigment gallstones.

Pigment gallstones are defined as any dark brown-to-black stone, consisting of calcium salts of bilirubin, phosphate, carbonate and other anions, and can be separated into carbonate- and noncarbonate-containing groups. Pigment stones predominate in the rural Orient, in cirrhosis, and in elderly United States patients undergoing cholecystectomy. Clinical associations include bile duct obstruction, stasis, and possibly hemolysis. Of pigment stones, 50% are radioopaque and account for two-thirds of all opaque stones. The concentrations of bile salts, phospholipids,, cholesterol, and total bilirubin in bile are similar to normal levels, but the concentration of unconjugated bilirubin is increased in the bile of some patients. Increased unconjugated bilirubin in bile may be caused by increased hydrolysis of excreted conjugated bilirubin. Unconjugated bilirubin is solubilized by bile salts, but the interaction is primarily nonmicellar. Ionized calcium and pH are important determinants of solubility. Sulfated glycoproteins, excreted in increased amounts in patients with cholelithiasis, may be the site of pigment stone precipitation because these compounds bind calcium salts tightly. E coli is frequently cultured from pigment stones in Japan but not in the United States; thus, bacterial beta-glucuronidase may be important in stone formation in Japan but probably not in the West. Stasis leads to increased calcium secretion and to increases in the concentration of sparingly soluble compounds that may then precipitate. Incomplete emptying of the gallbladder may result in the same concentration process. Unsaturated fats and chronic vagal stimulation cause pigment stone formation in animals. At present, surgery is the only treatment for pigment lithiasis.     

Beta-glucuronidase activity in the bile of gallstone patients both with and without duodenal diverticula

Patients with juxtapapillary duodenal diverticula have an increased occurrence of calcium bilirubinate gallstones. One possible hypothesis to explain this observation is enzymatic deconjugation of bilirubin conjugates in the bile. Beta-glucuronidase of human or bacterial origin may lead to deconjugation of the bilirubin glucuronides in bile. This, in turn, may increase the amounts of unconjugated, water-insoluble bilirubin which can precipitate as calcium bilirubinate, the main component of brown pigment stones. In this study we compared gallstone patients with and without duodenal diverticula treated with endoscopic papillotomy. Increased occurrence of bacteria producing beta-glucuronidase (p less than 0.01) and increased activity of bacterial beta-glucuronidase (pH 7.0) in the bile itself (p less than 0.01) were found in patients with duodenal diverticula. When the activity of the enzyme at pH 4.5, the optimum of the human enzyme, was measured, no such difference was found. The results support the hypothesis of bacterial glucuronidase as an etiologic factor in pigment gallstone disease in patients with duodenal diverticula. The high activity of bacterial enzyme found in the bile in some patients without diverticula suggests bacteria as an etiologic factor, independent of the presence of diverticula.